Compositions and methods for regulating angiogenesis and cholesterol and treating dyslipidemia, atherosclerosis, cancer and inflammatory conditions

ABSTRACT

The invention provides pharmaceutical compounds and formulations comprising nucleic acids and polypeptides for regulating (including upregulating or inhibiting) the expression of ApoA-1 Binding Protein (APOAIBP, AIBP, or AI-BP), and methods for making and using them. In alternative embodiments, APOAIBP-inhibiting pharmaceutical compositions and formulations of the invention are administered to an individual in need thereof in an amount sufficient to stimulate tissue revascularization, e.g., supporting or stimulating revascularization of heart tissue, e.g., after a cardiac ischemia. In alternative embodiments, pharmaceutical compositions and formulations of the invention that comprise APOAIBP nucleic acids and polypeptides or result in an increase in expression or activity of APOAIBP nucleic acids and polypeptides are administered to an individual in need thereof in an amount sufficient to treat, prevent, reverse and/or ameliorate a dyslipidemia, e.g., to treat, prevent, reverse and/or ameliorate conditions responsive to increasing cholesterol efflux from cells, including cardiovascular disease and atherosclerosis.

RELATED APPLICATIONS

This Patent Convention Treaty (PCT) International Application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/827,729, filed May 27, 2013. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes.

GOVERNMENT RIGHTS

This invention was made with government support under grants HL093767, HL055798, HL114734 and HL081862, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

TECHNICAL FIELD

This invention generally relates to medicine and cell biology. In particular, in alternative embodiments, the invention provides pharmaceutical compounds and formulations comprising nucleic acids and polypeptides for regulating (including upregulating or inhibiting) the expression of ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP), and methods for making and using them. In alternative embodiments, APOA1BP-inhibiting pharmaceutical compositions and formulations of the invention are administered to an individual in need thereof in an amount sufficient to stimulate tissue revascularization, e.g., supporting or stimulating revascularization of heart tissue, e.g., after a cardiac ischemia. In alternative embodiments, pharmaceutical compositions and formulations of the invention that comprise APOA1BP nucleic acids and polypeptides or result in an increase in expression or activity of APOA1BP nucleic acids and polypeptides are administered to an individual in need thereof in an amount sufficient to treat, prevent, reverse and/or ameliorate a dyslipidemia, e.g., to treat, prevent, reverse and/or ameliorate conditions responsive to increasing cholesterol efflux from cells, including cardiovascular disease and atherosclerosis. In alternative embodiments, pharmaceutical compositions and formulations of the invention that comprise APOA1BP nucleic acids and polypeptides or result in an increase in expression or activity of APOA1BP nucleic acids and polypeptides are administered to an individual in need thereof in an amount sufficient to treat, prevent, reverse and/or ameliorate neovascularization of a growth, an infection, a cancer or a tumor whose growth or survival depends on neovascularization. In alternative embodiments, the administered formulations and pharmaceutical compositions of the invention comprise an APOA1BP polypeptide or protein that is a human or a mammalian APOA1BP, or an AIBP1 or an AIBP2, or a recombinant, peptidomimetic or a synthetic APOA1BP, or a bioisostere of an ApoA-I Binding Protein.

BACKGROUND

Cholesterol is one of the building blocks of the cell and is absolutely required for normal cellular function, but its excess often leads to abnormal proliferation, migration, inflammatory responses and/or cell toxicity. Removal of cholesterol from the cells to apoA-I and to HDL, an apoA-I-containing lipoprotein, is mediated by ATP-binding cassette transporters (ABC), predominantly ABCA1 and ATP-binding cassette sub-family G member 1 (ABCG1) (1-3). Recent reports that ABCA1/G1-deficient hematopoietic cells tend to hyperproliferate (4-6) and that ABCG1 and HDL protect against endothelial dysfunction in hypercholesterolemic mice (7) suggest the importance of efficient cholesterol efflux in maintaining cellular function.

ApoA-I binding protein (AIBP) is a secreted protein discovered in a screen of proteins that physically associate with apoA-I (8). The human AIBP gene (APOA1BP) is located at 1q22. 1q21.2 on chromosome 1, which corresponds to the 1q21-q23 locus for familial combined hyperlipidemia, a common, multifactorial and heterogeneous dyslipidemia predisposing to premature coronary artery disease (9). This chromosomal location of the gene and the protein binding to apoA-I, the primary apolipoprotein involved in removal of cholesterol from cells, prompted the hypothesis that AIBP is involved in cholesterol efflux (8, 10). However, a function of AIBP in cholesterol metabolism has not been experimentally tested.

SUMMARY

In alternative embodiments, the invention provides formulations and pharmaceutical compositions comprising:

(a) an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP) polypeptide or peptide, or a compound or composition that increases expression or activity of a APOA1BP polypeptide or nucleic acid, or a polypeptide or peptide having a APOA1BP activity,

wherein optionally the polypeptide or peptide having a APOA1BP activity, or the APOA1BP polypeptide or peptide, comprises a recombinant, a synthetic, a peptidomimetic or an isolated polypeptide or peptide having a APOA1BP activity or APOA1BP polypeptide or peptide; or

(b) the formulation or pharmaceutical composition of (a), wherein the compound that increases expression or activity of a APOA1BP polypeptide is a nucleic acid that expresses a APOA1BP polypeptide or a polypeptide having a APOA1BP polypeptide activity,

and optionally a APOA1BP-stimulating compound or composition increases or stimulates (activates) the activity of a APOA1BP promoter or transcriptional regulatory sequence or motif

In alternative embodiments, the invention provides formulations and pharmaceutical compositions comprising:

(a) an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP)-inhibiting compound or composition;

(b) the formulation or pharmaceutical composition of (a), wherein the APOA1BP-inhibiting compound or composition inhibits or decreases the expression of a APOA1BP nucleic acid,

wherein optionally the nucleic acid inhibited is a APOA1BP gene or a APOA1BP message (mRNA),

and optionally the APOA1BP-inhibiting nucleic acid is an APOA1BP antisense oligonucleotide, or a APOA1BP-inhibiting miRNA or siRNA, and optionally the APOA1BP antisense oligonucleotide, or a APOA1BP-inhibiting miRNA or siRNA, comprises a morpholino oligonucleotide,

and optionally a APOA1BP-inhibiting compound or composition inhibits or decreases the activity of a APOA1BP promoter or transcriptional regulatory sequence or motif; or

(c) the formulation or pharmaceutical composition of (a), wherein the APOA1BP-inhibiting compound or composition inhibits or decreases the expression of a APOA1BP polypeptide,

wherein optionally the APOA1BP polypeptide-inhibiting compound is an anti-APOA1BP antibody or a APOA1BP-binding antibody or antibody-like fragment, a small molecule, a lipid or a carbohydrate.

In alternative embodiments of the formulations and pharmaceutical compositions of the invention:

the APOA1BP polypeptide or protein is a human or a mammalian APOA1BP, or a AIBP1 or a AIBP2, or a recombinant, peptidomimetic or a synthetic APOA1BP, or a bioisostere of an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP); or

the APOA1BP-inhibiting or APOA1BP-stimulating compound or composition is formulated:

(a) for administration in vitro or in vivo;

(b) for enteral or parenteral administration;

(c) as a liposome, a nanoparticle, or a nanoliposome;

(d) as a tablet, a pill, a capsule, a gel, a hydrogel, a geltab, a liquid, a powder, an emulsion, a lotion, an aerosol, a spray, a lozenge, an aqueous or a sterile or an injectable solution, an eye drop, or an implant; or

(e) intravenous injection, subcutaneous injection, intramuscular injection, inhalation, or intravitreal injection.

In alternative embodiments, the invention provides methods for stimulating cholesterol efflux from a cell, comprising:

(a) contacting the cell with an effective amount of a APOA1BP activity-increasing formulation or pharmaceutical composition comprising:

an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP) polypeptide or peptide, or a compound or composition that increases expression or activity of a APOA1BP polypeptide or nucleic acid, or a polypeptide or peptide having a APOA1BP activity, or a compound that increases expression or activity of a APOA1BP polypeptide is a nucleic acid that expresses a APOA1BP polypeptide or a polypeptide having a APOA1BP polypeptide activity,

wherein optionally the administration is in vitro, ex vivo or in vivo; or

(b) stimulating the expression of an exogenous or an endogenous nucleic acid expressing a polypeptide or peptide having an APOA1BP activity, or an APOA1BP polypeptide or peptide, wherein the nucleic acid comprises a gene or a message (mRNA), or an APOA1BP promoter or transcriptional regulatory sequence or motif;

and optionally the cell is an endothelial cell, or a human cell.

In alternative embodiments, the invention provides methods for:

inhibiting, reversing or reducing the rate of formation of lipid rafts or cholesterol-/sphingolipids membrane microdomains,

inhibiting receptor dimerization, or

increasing levels of high density lipoprotein (HDL) and/or improving HDL function, comprising:

(a) administering to an individual an effective amount of an APOA1BP activity increasing formulation or a pharmaceutical composition of the invention,

wherein optionally the formulation or pharmaceutical composition comprises a APOA1BP polypeptide or peptide having APOA1BP activity, a APOA1BP-stimulating polypeptide compound or composition, or a compound that increases expression or activity of a APOA1BP polypeptide or nucleic acid, wherein optionally the administration is ex vivo or in vivo,

wherein optionally the administration is in vitro, ex vivo or in vivo; or

(b) stimulating the expression of an exogenous or an endogenous nucleic acid expressing a polypeptide or peptide having an APOA1BP activity, or an APOA1BP polypeptide or peptide, wherein the nucleic acid comprises a gene or a message (mRNA), or an APOA1BP promoter or transcriptional regulatory sequence or motif,

and optionally the individual is a human.

In alternative embodiments, the invention provides methods for:

inhibiting or slowing vascular endothelial cell growth, or neovascularization, or angiogenesis,

treating, reversing, preventing or down-regulating an inflammatory response, an inflammation or a disease or condition caused by or creating an inflammation, or

treating, reversing, preventing or down-regulating a macular degeneration or a wet macular degeneration,

comprising:

(a) administering to an individual an effective amount of an APOA1BP activity increasing formulation or a pharmaceutical composition of the invention,

wherein optionally the formulation or pharmaceutical composition comprises a APOA1BP polypeptide or peptide having APOA1BP activity, a APOA1BP-stimulating polypeptide compound or composition, or a compound that increases expression or activity of a APOA1BP polypeptide or nucleic acid, wherein optionally the administration is ex vivo or in vivo,

wherein optionally the administration is in vitro, ex vivo or in vivo; or

(b) stimulating the expression of an exogenous or an endogenous nucleic acid expressing a polypeptide or peptide having an APOA1BP activity, or an APOA1BP polypeptide or peptide, wherein the nucleic acid comprises a gene or a message (mRNA), or an APOA1BP promoter or transcriptional regulatory sequence or motif,

and optionally the individual is a human,

and optionally the disease or condition caused by or creating an inflammation comprise an inflammatory bowel disease (IBD).

In alternative embodiments, the invention provides methods for:

inhibiting, preventing or slowing or reversing the growth of a vascularized tumor, a vascularized dysplastic tissue, a vascularized tissue, a vascularized growth, an infection dependent on neovascularization, or a cancer, comprising:

comprising:

(a) administering to an individual an effective amount of an APOA1BP activity increasing formulation or a pharmaceutical composition of the invention,

wherein optionally the formulation or pharmaceutical composition comprises a APOA1BP polypeptide or peptide having APOA1BP activity, a APOA1BP-stimulating polypeptide compound or composition, or a compound that increases expression or activity of a APOA1BP polypeptide or nucleic acid, wherein optionally the administration is ex vivo or in vivo,

wherein optionally the administration is in vitro, ex vivo or in vivo; or

(b) stimulating the expression of an exogenous or an endogenous nucleic acid expressing a polypeptide or peptide having an APOA1BP activity, or an APOA1BP polypeptide or peptide, wherein the nucleic acid comprises a gene or a message (mRNA), or an APOA1BP promoter or transcriptional regulatory sequence or motif, and optionally the individual is a human,

and optionally the cancer is a leukemia.

In alternative embodiments, the invention provides methods for:

treating, preventing, ameliorating or reversing the effects of a dyslipidemia, a hyperlipidemia, an atherosclerosis, a heart disease, a coronary artery disease, a heart attack, an ischemic event or a stroke:

comprising:

(a) administering to an individual an effective amount of an APOA1BP activity increasing formulation or a pharmaceutical composition of the invention,

wherein optionally the formulation or pharmaceutical composition comprises a APOA1BP polypeptide or peptide having APOA1BP activity, a APOA1BP-stimulating polypeptide compound or composition, or a compound that increases expression or activity of a APOA1BP polypeptide or nucleic acid, wherein optionally the administration is ex vivo or in vivo,

wherein optionally the administration is in vitro, ex vivo or in vivo; or

(b) stimulating the expression of an exogenous or an endogenous nucleic acid expressing a polypeptide or peptide having an APOA1BP activity, or an APOA1BP polypeptide or peptide, wherein the nucleic acid comprises a gene or a message (mRNA), or an APOA1BP promoter or transcriptional regulatory sequence or motif, and optionally the individual is a human.

In alternative embodiments, the invention provides methods for:

interfering with vascular endothelial growth factor receptor 2 (VEGRF2) (also known as kinase insert domain receptor, or KDR) dimerization or signaling, comprising:

(a) administering to an individual an effective amount of an APOA1BP activity increasing formulation or a pharmaceutical composition of the invention,

wherein optionally the formulation or pharmaceutical composition comprises a APOA1BP polypeptide or peptide having APOA1BP activity, a APOA1BP-stimulating polypeptide compound or composition, or a compound that increases expression or activity of a APOA1BP polypeptide or nucleic acid, wherein optionally the administration is ex vivo or in vivo,

wherein optionally the administration is in vitro, ex vivo or in vivo; or

(b) stimulating the expression of an exogenous or an endogenous nucleic acid expressing a polypeptide or peptide having an APOA1BP activity, or an APOA1BP polypeptide or peptide, wherein the nucleic acid comprises a gene or a message (mRNA), or an APOA1BP promoter or transcriptional regulatory sequence or motif,

and optionally the individual is a human.

In alternative embodiments, the invention provides methods for inhibiting, preventing or slowing or reversing:

a nonalcoholic steatohepatitis (NASH),

a diabetes,

an acute respiratory distress syndrome, or

a sepsis,

comprising:

(a) administering to an individual an effective amount of an APOA1BP activity increasing formulation or a pharmaceutical composition of the invention,

wherein optionally the formulation or pharmaceutical composition comprises a APOA1BP polypeptide or peptide having APOA1BP activity, a APOA1BP-stimulating polypeptide compound or composition, or a compound that increases expression or activity of a APOA1BP polypeptide or nucleic acid, wherein optionally the administration is ex vivo or in vivo,

wherein optionally the administration is in vitro, ex vivo or in vivo; or

(b) stimulating the expression of an exogenous or an endogenous nucleic acid expressing a polypeptide or peptide having an APOA1BP activity, or an APOA1BP polypeptide or peptide, wherein the nucleic acid comprises a gene or a message (mRNA), or an APOA1BP promoter or transcriptional regulatory sequence or motif, and optionally the individual is a human.

In alternative embodiments, the invention provides methods for:

promoting or accelerating revascularization,

promoting revascularization of a heart, an organ or a tissue, or

promoting revascularization of a heart, an organ or a tissue following an ischemic event or a stroke (e.g., promoting revascularization of a heart, an organ or a tissue post-ischemically), comprising:

administering a APOA1BP-inhibiting formulation or an APOA1BP activity-inhibiting pharmaceutical composition of the invention,

wherein optionally the APOA1BP-inhibiting compound or composition inhibits or decreases the expression of a APOA1BP nucleic acid,

and optionally the nucleic acid inhibited is a APOA1BP gene or a APOA1BP message (mRNA),

and optionally the APOA1BP-inhibiting nucleic acid is an APOA1BP antisense oligonucleotide, or a APOA1BP-inhibiting miRNA or siRNA,

and optionally a APOA1BP-inhibiting compound or composition inhibits or decreases the activity of a APOA1BP promoter or transcriptional regulatory sequence or motif;

and optionally the APOA1BP-inhibiting compound or composition inhibits or decreases the expression of a APOA1BP polypeptide,

and optionally the APOA1BP polypeptide-inhibiting compound is an anti-APOA1BP antibody or a APOA1BP-binding antibody or antibody-like fragment, a small molecule, a lipid or a carbohydrate.

In alternative embodiments, the invention provides kits comprising a formulation or a pharmaceutical composition of the invention, and optionally comprising instructions on practicing a method of the invention.

In alternative embodiments, the invention provides nanoparticles, nanolipoparticles, vesicles or liposomal membranes comprising a formulation or a pharmaceutical composition of the invention.

In alternative embodiments, the invention provides use of a formulation or a pharmaceutical of the invention, or a nanoparticle, nanolipoparticle, vesicle or liposomal membrane of the invention, or a combination thereof, in the manufacture of a medicament.

In alternative embodiments, the invention provides use of a formulation or a pharmaceutical of the invention, or a nanoparticle, nanolipoparticle, vesicle or liposomal membrane of the invention, in the manufacture of a medicament for interfering with vascular endothelial growth factor receptor 2 (VEGRF2 or KDR) dimerization or signaling.

In alternative embodiments, the invention provides use of a formulation or a pharmaceutical of the invention, or nanoparticle, nanolipoparticle, vesicle or liposomal membrane of the invention, in the manufacture of a medicament for promoting or accelerating revascularization, or promoting revascularization of the heart, or promoting revascularization of the heart following an ischemic event (promoting post-ischemic vascularization).

In alternative embodiments, the invention provides use of a formulation or a pharmaceutical of the invention, or nanoparticle, nanolipoparticle, vesicle or liposomal membrane of the invention, in the manufacture of a medicament for treating, preventing, ameliorating or reversing the effects of a dyslipidemia, hyperlipidemia, atherosclerosis or coronary artery disease.

In alternative embodiments, the invention provides use of a formulation or a pharmaceutical of the invention, or nanoparticle, nanolipoparticle, vesicle or liposomal membrane of the invention, in the manufacture of a medicament for inhibiting or slowing or reversing the growth of a vascularized tumor, dysplastic tissue, tissue or growth, or a cancer.

In alternative embodiments, the invention provides use of a formulation or a pharmaceutical of the invention, or nanoparticle, nanolipoparticle, vesicle or liposomal membrane of the invention, in the manufacture of a medicament for inhibiting or slowing vascular endothelial cell growth, or neovascularization, or angiogenesis.

In alternative embodiments, the invention provides use of a formulation or a pharmaceutical of the invention, or nanoparticle, nanolipoparticle, vesicle or liposomal membrane of the invention, in the manufacture of a medicament for inhibiting, reversing or reducing the rate of formation of lipid rafts or cholesterol-/sphingolipids membrane microdomains, or increasing levels of high density lipoprotein (HDL) and/or improving HDL function.

In alternative embodiments, the invention provides use of a formulation or a pharmaceutical of the invention, or nanoparticle, nanolipoparticle, vesicle or liposomal membrane of the invention, in the manufacture of a medicament for stimulating cholesterol efflux from a cell.

In alternative embodiments, the invention provides use of a formulation or a pharmaceutical of the invention, or nanoparticle, nanolipoparticle, vesicle or liposomal membrane of the invention, in the manufacture of a medicament for inhibiting, preventing or slowing or reversing: a nonalcoholic steatohepatitis (NASH), a diabetes, an acute respiratory distress syndrome, or a sepsis.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings set forth herein are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

FIG. 1 illustrates the role of AIBP in zebrafish angiogenesis:

FIG. 1(A) illustrates images of one-cell stage fli1:EGFP zebrafish embryos that were injected with 8 ng of either control morpholino oligonucleotide (MO) or AIBP2 antisense morpholino oligonucleotides (MO) targeting ATG translation site, and the images of segmental arteries (SeA) (images 1-2) and of subintestinal veins (SIV) (images 3-4) were captured 3 days after MO injection and arrows point to dysregulated sprouts;

FIG. 1(B) graphically illustrates the numbers of embryos with abnormal angiogenesis resulting from injection of control or AIBP2 MO;

FIG. 1(C) illustrates images of one-cell stage fli1:nEGFP embryos that were injected with 8 ng of either control MO or AIBP2 MO;

FIG. 1(D) graphically illustrates the quantification of endothelial cell (EC) nuclei in images of segmental arteries (SeA);

FIG. 1(E) illustrates the tissue distribution of AIBP2 mRNA in zebrafish embryos at the 20 minutes (min) post fertilization, the 12 hours post fertilization (hpt) and the 24 hours post fertilization (hpf) developmental stages;

FIG. 1(F) illustrates images of a mosaic expression analysis of endothelial cell (EC) branching in wild type (WT) and AIBP2 knockdown (KD) embryos, arrowheads indicate aberrant ectopic branches/sprouts in the AIBP2 KD recipients; and,

FIG. 1(G) graphically illustrates the numbers of ectopic branches/filopodial projections per SeA;

as described in detail in Example 1, below.

FIG. 2 illustrates images and data showing upregulated markers of endothelial cell (EC) proliferation and angiogenesis in AIBP2 morphants:

FIG. 2(A) graphically illustrates the expression of genes involved in angiogenesis, including endothelium-specific receptor tyrosine kinase 2 (Tie2), kinase insert domain receptor (KDR), Fetal Liver Kinase 1 (Flk1) and Friend leukemia integration 1 transcription factor (FLI1);

FIG. 2(B) illustrates whole embryo in situ hybridization (WISH) analysis of genes involved in angiogenesis, including endothelium-specific receptor tyrosine kinase 2 (Tie2), Fetal Liver Kinase 1 (Flk1), Fms-related tyrosine kinase 4 (Flt4), neurogenic locus notch homolog protein 3 (Notch3), and Delta like ligand 4 (Dll4);

FIG. 2(C) illustrates immunoblot images of Akt (also known as Protein Kinase B (PKB)) and extracellular-signal-regulated kinases (ERK) ½ phosphorylation;

FIG. 2(D), FIG. 2(E) and FIG. 2(F) illustrate free cholesterol levels in AIBP2 morphants, where FIG. 2(D) illustrates fluorescence images of 24 hours post fertilization (hpf) control and AIBP2 morphants stained with filipin to detect free cholesterol in embryos, and FIG. 2(E) graphically illustrates the intensities of filipin fluorescence images, and FIG. 2(F) graphically illustrates free cholesterol levels determined by gas chromatography (GC), in one-cell stage embryos injected with control or AIBP2 MO;

as described in detail in Example 1, below.

FIG. 3 illustrates images and data showing the effect of AIBP on cholesterol efflux:

FIG. 3(A) illustrates different fractions of lipoproteins run on SDS-PAGE and immunoblotted with indicated antibodies;

FIG. 3(B) illustrates immunoprecipitated and immunoblotted purified human ApoA-I mixed with His-tagged AIBP2 or hAIBP;

FIG. 3(C) graphically illustrates cholesterol efflux to HDL3, where human umbilical vein endothelial cells (HUVEC) cells were preloaded with 3H-cholesterol and then incubated with media, HDL3, HDL3+His-AIBP, or HDL3+His-DUB, and the media and cell lysates were collected and 3H counts measured;

FIG. 3(D) graphically illustrates the kinetics of cholesterol efflux from HUVEC to HDL3, with the experimental conditions as in FIG. 3C;

FIG. 3(E) illustrates images showing the effects of hAIBP and HDL3 on EC tube formation, where HUVEC were preincubated with HDL3, His-hAIBP, or HDL3+His-hAIBP, cells were then seeded on MATRIGEL™ (BD Biosciences) in the presence or absence of VEGF, and imaged following a 12 hour incubation;

FIG. 3(F) (upper graph) graphically illustrates data showing the effect of ATP-binding cassette sub-family G member 1 (ABCG1) knockdown on hAIBP mediated-cholesterol efflux, where HUVEC were transfected with control or ABCG1 siRNA, and (lower gel) illustrates by western blot ABCG1 knockdown;

as described in detail in Example 1, below.

FIG. 4 illustrates images and data showing the role of ABCA1/G1 in angiogenesis and in mediating effects of AIBP:

FIG. 4(A) and FIG. 4(D) illustrate images of segmental arteries (SeA) (A) and SIV (D), and FIG. 4(B), FIG. 4(C), FIG. 4(E) and FIG. 4(F) graphically illustrate: angiogenic defects in ABCA1/G1 morphants, where one-cell stage embryos were injected with a control MO, AIBP2 MO, or ABCA1 MO+ABCG1 MO, and 3 days later the morphants were analyzed by confocal microscopy; the graphs show number and length of segmental arteries (SeA) ectopic branches (FIG. 4B and FIG. 4C) and SIV ectopic sprouts (FIG. 4E and FIG. 4F) from 5 to 12 embryos;

FIG. 4(B) illustrates images of, and FIG. 4(H) graphically illustrates, the rescue of the effect of AIBP2 overexpression in ABCA1/G1 morphants, where FIG. 4(G) illustrates confocal microscope images of one cell stage embryos injected with myog-AIBP2-mCherry plasmid, myog-AIBP2-mcherry plasmid together with ABCA1/G1 MO, or myog-mCherry, and two days later, the embryos were analyzed by confocal microscope;

FIG. 4(I) schematically illustrates a diagram representing the hypothesis that somite expression of AIBP2 accelerates lateral cholesterol efflux from growing endothelial cell (EC) and thereby prevents its ectopic sprouting/branching;

as described in detail in Example 1, below.

FIG. 5 (or FIG. S1A, of Example 1) illustrates images aligning and comparing AIBP protein amino acid sequences of human (SEQ ID NO:43), mouse (SEQ ID NO:44), zebrafish (SEQ ID NO:45), Drosophila (SEQ ID NO:46), and a consensus sequence (SEQ ID NO:47), showing a conservation of AIBP proteins across different species. AIBP protein sequences were retrieved from ENZEMBL™ (EMBL-EBI; Wellcome Trust Sanger Institute) and were compared using VECTOR NTI™ software (Invitrogen); as described in detail in Example 1, below.

FIG. 6 (or FIG. S1B, of Example 1) illustrates images aligning and comparing the isoforms of zebrafish AIBP; comparisons were performed using VECTOR NTI™ software; as described in detail in Example 1, below.

FIG. 7 (or FIG. S2, of Example 1) illustrates images showing dysregulated SeA and SIV angiogenesis in AIBP2 morphants (splicing site); Onecell stage fli1:EGFP zebrafish embryos were injected with 8 ng of either control MO or AIBP2 MO2 targeting a splicing site; the images of SeA (FIG. 7A and FIG. 7B) and SIV (FIG. 7C) were captured 3 days after MO injection; arrows point to dysregulated sprouts; scale: 50 m. FIG. 7D left schematic illustrates a diagram showing targeted splicing site and FIG. 7D right image illustrates a gel showing corresponding PCR products; PF and PR indicate the primers used for the amplification; as described in detail in Example 1, below.

FIG. 8 (or FIG. S3, of Example 1) illustrates tables showing selected results from microarray analysis of 12 hours post fertilization (hpf) AIBP2 morphants; one cell stage embryos were injected with either control or AIBP2 MO; twelve hours later, total RNA of injected embryos were extracted and analyzed in a microarray; as described in detail in Example 1, below.

FIG. 9(A) (or FIG. S4, of Example 1) illustrates an image of an SDS gel probed with antibodies showing the specificity of AIBP2 antibody; a polyclonal antibody against zebrafish AIBP2 (guinea pig post-immune serum) was used to probe zebrafish homogenates; fifteen, 10 and 5 μl of the homogenates were loaded on an SDS gel and probed with the antibody, preincubated with 10 μg of either ubiqitine (Ub) or AIBP2 (both recombinant proteins were produced from the same expression vector and under the same conditions); the blots were visualized with an anti-guinea pig IgG conjugated with HRP and a chemiluminescent substrate; as described in detail in Example 1, below.

FIG. 9(B) (or FIG. S5, of Example 1) illustrates an image of an agarose gel electrophoresis of human lipoprotein fractions separated by ultracentrifugation; DUB, deubiquitinase; Sta, human standard plasma; LPDS, lipoprotein-deficient serum; as described in detail in Example 1, below.

FIG. 10(A) (or FIG. S6, of Example 1) illustrates an image of an SDS gel probed with antibody probes showing the effect of AIBP on Akt and ERK1/2 phosphorylation in HUVEC; where HUVEC were preincubated with 50 μg/ml HDL3, or 50 μg/ml HDL3+0.2 μg/ml His-AIBP for 4 hours, followed by a 20 min stimulation with 50 ng/ml VEGF. Cells were lysed, and the lysates run on SDS-PAGE and probed as indicated; as described in detail in Example 1, below.

FIG. 10(B) (or FIG. S7, of Example 1) illustrates images of SIV sprouting in AIBP2 and ABCA1/G1 morphants; where FIG. 10(B)-A, FIG. 10(B)-B, and FIG. 10(B)-C, illustrate images of one-cell stage embryos of Tg(fli1:negfp)_(y7) transgenic zebrafish in which EC nuclei are fluorescent green, were injected with 8 ng of control MO, 8 ng AIBP2 MO, or 4 ng ABCA1 MO+4 ng ABCG1 MO, and the embryos were analyzed at 3 dpf; red arrows indicate excessive cell proliferation in the ectopic sprouts; scale, 20 μm. FIG. 10(B)-D graphically illustrates the numbers of cells per SIV sprout were quantified in 6-15 embryos per group. **, p<0.01; as described in detail in Example 1, below.

FIG. 11 (or FIG. S8, of Example 1) illustrates an image and graphs showing free cholesterol levels in ABCA1/G1 morphants: FIG. 11(A) illustrates representative images of 24 hpf control and ABCA1/G1 morphants stained with filipin to detect free cholesterol; note the yolks are artificially masked; FIG. 11(B) graphically illustrates data showing the intensity of filipin fluorescence in images shown in panel A (FIG. 11(A)) (without yolks) was quantified in 8-10 embryos per group. *, p<0.05; FIG. 11(C) graphically illustrates data showing free cholesterol levels of one-cell stage embryos which were injected with control or AIBP2 MO, and at 24 hpf, the trunk area (without yolk and head) was dissected and total lipid was extracted; free cholesterol levels were determined by GC; the cholesterol levels were normalized to the protein content and then to the values in control MO embryos; 50 to 70 embryos were pooled for each sample; the data are from 3-4 independent experiments; as described in detail in Example 1, below.

FIG. 12 (or FIG. 1, of Example 2) in images and graphics shows the role of AIBP in cholesterol efflux from endothelial cell (EC) and in vitro angiogenesis:

FIG. 12a graphically illustrates data showing the effect of ATP-binding cassette sub-family G member 1 (ABCG1) knockdown on hAIBP mediated-cholesterol efflux;

FIG. 12b illustrates images showing the effect of hAIBP and HDL3 on EC tube formation where HUVEC were preincubated with HDL3, hAIBP, or HDL3+hAIBP for 4 hours;

FIG. 12c upper image illustrates images showing the requirement for ABCG1 in hAIBP inhibition of angiogenesis, where HUVEC were transfected with control or ABCG1 siRNA and used in an EC tube formation assay, and FIG. 12c lower graph illustrates this data;

FIG. 12d illustrates images showing the effect of hAIBP and HDL3 on lipid rafts;

FIG. 12e illustrates images of probed SDS-PAGE showing the effect of hAIBP on VEGF signaling;

FIG. 12f upper illustrates images showing an aortic ring angiogenesis assay wherein the aorta was isolated from a C57BL6 mouse and sliced into 1 mm wide rings, and the aortic rings were embedded in MATRIGEL™, and HEK293 cells expressing zAibp2 or hAIBP were inserted approximately 0.5 mm away from the aortic ring, and the plates were incubated with 10 ng/ml VEGF for 7 days; FIG. 12f lower illustrates immunoblots showing expression of hAIBP and zAibp2 in HEK293 cells;

as described in detail in Example 2, below.

FIG. 13 (or FIG. 2, of Example 2) in images and graphics shows the effect of AIBP deficiency on zebrafish cholesterol metabolism and angiogenesis:

FIG. 13a illustrates by image, and FIG. 13b and FIG. 13c graphically illustrate, free cholesterol levels in zaibp2 morphants:

FIG. 13a illustrates an image of one-cell stage AB zebrafish embryos injected with either control MO or zaibp2 MO targeting ATG translation site, and twenty four hpf control and zaibp2 morphants were fixed and stained with filipin to detect free cholesterol in embryos;

FIG. 13b graphically illustrates data where the intensities of filipin fluorescence in images shown in panel FIG. 13A were quantified in 12 to 15 embryos per group;

FIG. 13c graphically illustrates data of free cholesterol levels as determined by GC, where at 24 hpf, the trunk area was dissected and total lipids were extracted, and the cholesterol levels were normalized to the protein content and then to the values in control MO embryos;

FIG. 13d , FIG. 13e and FIG. 13f illustrate images showing the angiogenic defects in zaibp2 morphants, where one-cell stage Tg(fli1:egfp)y1 zebrafish embryos were injected with either control MO or zaibp2 MO targeting ATG translation site:

FIG. 13d illustrates low magnification bright field images showing the area of fluorescent imaging of SeA (right, or white box) and SIV (left, or red box), in a control (upper image) and a zaibp2 morphant (lower image);

FIG. 13e illustrates images of SeA in 30 hpf embryos (top row), and SeA (middle and panel images) and of SIV (lower panel images) in 3 dpf embryos, where the arrows point to dysregulated sprouts, for control and zaibp2 morphant;

FIG. 13f graphically illustrates a quantification of the number of embryos with normal and abnormal angiogenesis (SeA with ectopic branching) resulting from injection of control or zaibp2 MO;

as described in detail in Example 2, below.

FIG. 14 (or FIG. 3, of Example 2) in images and graphics shows the effect of AIBP deficiency and ectopic overexpression on zebrafish angiogenesis and VEGF signaling:

FIG. 14a illustrates images of tissue distribution of zaibp2 mRNA in zebrafish embryo, where embryos at different developmental stages were fixed and whole mount in situ hybridization (WISH) was performed with an antisense zaibp2 probe;

FIG. 14b left panels illustrates images of a mosaic expression analysis of EC branching in WT and zaibp2 knockdown embryos; red arrowheads indicate aberrant ectopic branches/sprouts; and FIG. 14b right graph shows data quantifying the number of ectopic sprouts per SecA;

FIG. 14c illustrates images showing that the ectopic expression of zAibp2 inhibits SeA growth: one-cell stage Tg(fli1:egfp)y1 embryos were injected with DNA constructs for myog:Ga14, DsRed:10×UAS:zAibp2 or myog:Ga14, DsRed:10×UAS expression; images were captured at 30 hpf; arrowheads point to aberrant SeA growth close to the sites of DsRed (and zAibp2) expression;

FIG. 14d graphically illustrates data showing the expression of genes involved in angiogenesis; total RNA was isolated from 24 hpf control and zaibp2 morphants and qRT-PCR was performed;

FIG. 14e illustrates images of SDSPAGE and immunoblots showing phosphorylation of signaling proteins: lysates of 24 hpf control and control MO, zaibp2 and abca1/g1 (abca1 MO+abcg1 MO); morphants were separated on SDSPAGE and immunoblotted as indicated;

as described in detail in Example 2, below.

FIG. 15 (or FIG. 4, of Example 2) in images and graphics shows the role of Abca1 and Abcg1 in angiogenesis and in mediating effects of Aibp:

FIG. 15a left panel images illustrate images showing the angiogenic defects in abca1/g1 morphants; one-cell stage embryos were injected with control MO, zaibp2 MO, or abca1 MO+abcg1 MO; and images of SeA (30 hpf) and SIV (3 dpf) are shown; FIG. 15a right graphs show numbers and of SeA ectopic branches and numbers and length of SIV ectopic sprouts from 5 to 12 embryos;

FIG. 15b left panel images illustrate images showing rescue of the effect of zAibp2 overexpression in abca1/g1 morphants; one-cell stage embryos were injected with myog:zAibp2-mCherry plasmid, myog:zAibp2-mCherry plasmid together with abca1/g1 MO, or myog:mCherry plasmid; the arrow points to an aberrant SeA at the site of zAibp2-mCherry expression; FIG. 15b right graphs show data quantifying abnormal SeA formation in 8 to 16 embryos per group;

as described in detail in Example 2, below.

FIG. 16 (or FIG. 1, of Example 3) in images and graphics shows the role of AIBP in cholesterol efflux from EC and in vitro angiogenesis:

FIG. 16a graphically illustrates hAIBP mediated-cholesterol efflux and effect of ABCG1 knockdown; HUVEC were transfected with control or ABCG1 siRNA, preloaded with ³H-cholesterol and incubated for 1 hour with 50 μg/ml HDL₃ in the presence or absence of 0.2 μg/ml hAIBP; and, efflux was measured as the ³H counts in the medium divided by the sum of ³H counts in the medium and the cell lysate;

FIG. 16b and FIG. 16c graphically illustrate the effect of hAIBP on HDL₃ binding to HUVEC: HUVEC were incubated on ice with the indicated concentration of biotinylated HDL₃ (b-HDL₃), in the presence or absence of hAIBP (at a 0.1:50 w/w hAIBP:HDL₃ ratio) and 40× excess of unlabeled HDL:

FIG. 16d illustrates images of the effect of hAIBP and HDL₃ on EC tube formation; HUVEC were preincubated with or without HDL₃+hAIBP for 4 hours; cells were then seeded on MATRIGEL™, in the presence or absence of VEGF, and imaged following a 12-hour incubation;

FIG. 16e graphically illustrates data from the experiment of FIG. 16 d;

FIG. 16f graphically illustrates data showing the requirement for ABCG1 in hAIBP inhibition of angiogenesis; HUVEC were transfected with control or ABCG1 siRNA and assayed as in FIG. 16 d;

FIG. 16g left images illustrates images a mouse aortic ring angiogenesis assay: aortic rings from C57BL6 and Abcg1^(−/−) mice were embedded in MATRIGEL™; HEK293 cells transiently expressing mCherry, zAibp2 or hAIBP were inserted approximately 0.5 mm away from the aortic ring, and the plates were incubated with 10 ng/ml VEGF for 7 days, and the images show the edge of the aortic rings facing the HEK293 cell clusters, and FIG. 16g right images illustrate immunoblots showing expression of hAIBP and zAibp2, both detected with a Flag tag antibody, and mCherry in HEK293 cells;

FIG. 16h graphically illustrates data showing the length of aortic ring sprouts;

as described in detail in Example 3, below.

FIG. 17 (or FIG. 2, of Example 3) in images and graphics show the effect of AIBP on HUVEC lipid rafts, VEGFR2 localization, dimerization and signaling:

FIG. 17a illustrates images of the effect of hAIBP and HDL₃ on lipid rafts; HUVEC were preincubated with HDL₃, hAIBP, or HDL₃+hAIBP for 4 hours, cells were stained for nuclei (blue, DAPI) and for lipid rafts (red, cholera toxin B (CTB) and anti-CTB antibody;

FIG. 17b graphically illustrates data from the experiment of FIG. 17 a;

FIG. 17c graphically illustrates data showing the effect of hAIBP and HDL₃ on caveolin-1 and VEGFR2 surface localization: HUVEC were incubated with hAIBP and/or HDL₃ as in FIG. 17a , fixed and stained with antibodies to caveolin-1 and VEGFR2, and images were captured using TIRF microscopy and Pearson's coefficient was calculated to assess surface colocalization of VEGFR2 with caveolin-1;

FIG. 17d illustrates images of SDS-PAGE and Western blots showing VEGFR2 and caveolin-1 localization to lipid rafts: HUVEC were incubated with cholesterol-MβCD for six hours, followed by a 1 hour incubation with or without HDL₃+hAIBP, or a 30 min incubation with MβCD, and HUVEC lysates were separated into lipid rafts and non-lipid rafts fractions by ultracentrifugation, run on SDS-PAGE and blotted with VEGFR2 and caveolin-1 antibodies;

FIG. 17e illustrates images of SDS-PAGE and Western blots showing the effect of hAIBP and HDL₃ on VEGFR2 dimerization: HUVEC were preincubated with HDL₃ and/or hAIBP as in FIG. 16a , followed by a 20 min stimulation with VEGF, cells were treated with a crosslinking reagent, lysed and immunoprecipitated with a VEGFR2 antibody, and monomers and crosslinked dimers of VEGFR2 were visualized on Western blot;

FIG. 17f illustrates images showing the effect of hAIBP and HDL₃ on VEGFR2 endocytosis: HUVEC were preincubated with or without HDL₃+hAIBP for 4 hours, then stimulated with VEGF for 20 min, fixed and stained with antibodies to VEGFR2 (red) and the early endosome marker EEA-1 (green); yellow and white arrows point to the surface and endosomal localization of VEGFR2; and red dotted line traces cell contour;

FIG. 17g illustrates images of SDS-PAGE and Western blots showing the effect of hAIBP and HDL₃ on VEGFR2 signaling: HUVEC were preincubated with HDL₃ and/or hAIBP as in 2a, followed by a 20 min stimulation with VEGF, and total cell lysates were run on SDS-PAGE and probed as indicated;

as described in detail in Example 3, below.

FIG. 18 (or FIG. 3, of Example 3) in images and graphics show the effect of Aibp deficiency on zebrafish cholesterol, membrane lipid order, Vegfr2 signaling and angiogenesis:

FIG. 18a illustrates images of zebrafish embryos showing the tissue distribution of zaibp2 mRNA in the embryos; embryos at 24 hpf were fixed and WISH was performed with antisense myod and zaibp2 probes;

FIG. 18b and FIG. 18c illustrates free cholesterol levels in zaibp2 morphants:

FIG. 18b illustrates images of zebrafish embryos injected with either control MO or zaibp2 MO; twenty four hpf control and zaibp2 morphants were stained with filipin to detect free cholesterol in embryos;

FIG. 18c graphically illustrates data of free cholesterol levels determined by gas chromatography (GC), where zebrafish embryos at 24 hpf, the trunk area (without yolk) was dissected, total lipids extracted, and free cholesterol levels determined;

FIG. 18d illustrates images showing the effect of zaibp2 MO on SeA membrane lipid order; Tg(flk1:ras-cherry)^(s896) embryos were injected with control or zaibp2 MO as in 3b and at 24 hpf were stained with 5 μM Laurdan; in the same embryos, confocal images of mCherry fluorescence (bottom images) and the multiphoton images of Laurdan fluorescence were captured, and the multiphoton results (top row images) are displayed as pseudocolored GP (generalized polarization, a measure of the membrane lipid order) images, cropped to show only the vasculature, i.e. mCherry-positive areas;

FIG. 18e graphically illustrates data of GP values in the areas corresponding to tip and stalk cells of growing SeA and the dorsal aorta (DA) as indicated in FIG. 18 d;

FIG. 18f illustrates images of SDS-PAGE and immunoblots showing phosphorylation of signaling proteins: lysates of 24 hpf control (8 ng control MO), zaibp2 (8 ng zaibp2 MO) and abca1/g1 (4 ng abca1 MO+4 ng abcg1 MO) morphants were separated on SDS-PAGE and immunoblotted as indicated;

FIG. 18g illustrates images of zebrafish embryos showing angiogenic defects in zaibp2 morphants: one-cell stage Tg(fli1: egfp)^(y1) zebrafish embryos were injected with 8 ng of either control or zaibp2 MO; the images are of SeA in 30 hpf embryos (top row), and SeA (middle row) and of SIV (bottom row) in 3 dpf embryos; arrows point to dysregulated sprouts;

FIG. 18h graphically illustrates data showing the quantification of the number of embryos with normal and abnormal angiogenesis (SeA with ectopic branching); The abnormal angiogenesis was partially rescued by co-injection of 40 pg of zaibp2 mRNA lacking the MO targeting site;

as described in detail in Example 3, below.

FIG. 19 (or FIG. 4, of Example 3) in images and graphics show the effects of Aibp and Abca1/Abcg1 deficiency on zebrafish angiogenesis:

FIG. 19a illustrates images of zebrafish embryos using a mosaic expression analysis of EC branching in control, zaibp2 and abca1/abcg1 knockdown embryos; at 4 hpf, cells were isolated from donor embryos and transplanted into recipient embryos; recipient embryos were analyzed at 3 dpf; arrows point to aberrant ectopic branches/sprouts;

FIG. 19b graphically illustrates data showing numbers of ectopic branches/filopodial projections per SeA;

FIG. 19c illustrates images of angiogenic defects in abca1/abcg1 morphants: one-cell stage embryos were injected with 8 ng of control MO, 8 ng zaibp2 MO, or 4 ng abca1 MO+4 ng abcg1 MO; images of SeA (30 hpf) and SIV (3 dpf) are shown;

FIG. 19d illustrates images of zebrafish embryos where knockdown of abca1/g1 cancels the effect of zAibp2 overexpression: one-cell stage embryos were injected with 2 nl of 100 ng/μl myog:zaibp2-mCherry, myog:zaibp2-mCherry+abca1/g1 MO, or myog:mCherry; arrow points to an aberrant SeA at the site of zAibp2-mCherry expression;

FIG. 19e graphically illustrates data showing abnormal SeA formation (as a percent of SeA sprouting) as quantified in 8 to 16 embryos per group;

as described in detail in Example 3, below.

FIG. 20 (or supplementary FIG. 1, Example 3) schematically illustrates the hypothesis that expression of Aibp2 in somites (tan-shaded areas) but not in the inter-somitic space accelerates cholesterol efflux from EC to HDL; because cholesterol removal from the plasma membrane reduces lipid rafts and inhibits Vegfr2 signaling, the tip cell is guided in the direction where Aibp2 is least present; in aibp2 knockdown embryos (right image) defective cholesterol efflux leads to a higher abundance of lipid rafts (depicted as dark green boundaries of EC in right-hand panel) and enhanced Vegfr2 signaling, which result in misguided growth and ectopic branching of SeA, as further discussed in Example 3, below.

FIG. 21a and FIG. 21b (or Supplementary FIG. 2, Example 3):

FIG. 21a illustrates images of siRNA in ABCG1 knockdown in HUVEC: the blot confirms the ABCG1 knockdown in cells used in the experiments of FIG. 16 a;

FIG. 21b graphically illustrates data showing the kinetics of cholesterol efflux from HUVEC to HDL₃: HUVEC were preloaded with ³H-cholesterol and then incubated for 2, 4 or 6 hours with 50 μg/ml HDL₃ in the presence or absence of 0.2·g/ml human AIBP or 0.2·g/ml deubiquitinase (DUB, a non-relevant protein of the same molecular weight as AIBP); The cholesterol efflux was measured as described Example 3, below; note that AIBP in absence of HDL₃ did not induce cholesterol efflux from HUVEC. Mean±SD; n=3. *, p<0.05; **, p<0.01;

FIG. 21c (or Supplementary FIG. 3, Example 3): graphically illustrates data of human AIBP binding to HUVEC: HUVEC were incubated on ice with the indicated concentrations of biotinylated AIBP (b-AIBP), in the presence or absence of HDL₃ (at a 2:1 w/w AIBP:HDL₃ ratio), and the b-AIBP binding was determined by a chemiluminescent assay; a non-linear regression fit of both binding curves yielded K_(d)=(1.1±0.4)×10⁻⁷ M for AIBP/HUVEC binding.

FIG. 22A (or Supplementary FIG. 4, Example 3): illustrates images showing the effect of human AIBP and HDL₃ on EC tube formation: this figure is an extension of FIG. 16d , to illustrate the results shown on the graph of FIG. 16e ; for that reason, the top and bottom row images in this figure are identical to the images in FIG. 16d : HUVEC were preincubated with 50 μg/ml HDL₃, 0.1 μg/ml AIBP, or 50 μg/ml HDL₃+0.1 μg/ml AIBP, and cells were then seeded on MATRIGEL™, in the presence or absence of 20 ng/ml VEGF, and imaged following a 12 hour incubation.

FIG. 22B and FIG. 22C (or Supplementary FIG. 5, Example 3): FIG. 22B illustrates images showing the effect of MβCD and cholesterol-MβCD on EC tube formation: HUVEC were preincubated with 10 mM MβCD for 30 min or 20 μg/ml cholesterol-MβCD for 4 hours; cells were then seeded on MATRIGEL™ in the presence or absence of 20 ng/ml VEGF, and imaged following a 6 hour incubation; scale, 100 μm. Mean±SD; n=3. *, p<0.05; ***, p<0.001 vs. VEGF/control; FIG. 22C graphically illustrates data of EC tube formation length in control, MβCD and cholesterol-MβCD samples;

FIG. 23a and FIG. 23b (or Supplementary FIG. 6a and FIG. 6b , Example 3): illustrate total internal reflection fluorescence (TIRF) microscopy images of the effect of human AIBP and HDL₃ on caveolin-1 and VEGFR2 surface localization:

FIG. 23a illustrates the results shown on the graph of FIG. 17c : HUVEC were treated as in FIG. 17a , fixed and stained with antibodies to caveolin-1 (green) and VEGFR2 (red), and images were captured using TIRF microscopy; scale, 10 μm.

FIG. 23b illustrates the negative control, where HUVEC were stained with a mouse IgG1 isotype (replacing the mouse caveolin-1 antibody) and a rabbit anti-GFP antibody (replacing the VEGFR2 antibody), followed by secondary antibodies, as further discussed in Example 3, below.

FIG. 24A (or Supplementary FIG. 6c , Example 3): graphically illustrates the non-specific green and red signals of FIG. 23a and FIG. 23b , showing that there was no colocalization of non-specific green and red signals as shown with Pearson's coefficient calculations, as further discussed in Example 3, below.

FIG. 24B (or Supplementary FIG. 7, Example 3): graphically illustrates the effect of human AIBP and HDL₃ on VEGFR2 and caveolin-1 localization to lipid rafts. This supplementary figure is the quantification of VEGFR2 and caveolin-1 in the lipid raft fraction (LR) shown in FIG. 2d . Equal protein amounts of each sample were loaded on gel. Mean±SD. The experiment was repeated 5 times for HDL₃+AIBP and 3 times for MβCD treatments; *, p<0.05; ***, p<0.001, as further discussed in Example 3, below.

FIG. 25 (or Supplementary FIG. 8, Example 3): graphically illustrates the effect of human AIBP and HDL₃ on VEGFR2 dimerization: this figure is the quantification of the VEGFR2 dimerization experiment shown in FIG. 17e , calculated as dimer/(monomer+dimer). Mean±SD (n=3); *, p<0.05, as further discussed in Example 3, below.

FIG. 26 (or Supplementary FIG. 9, Example 3): graphically illustrates the effect of human AIBP and HDL₃ on VEGFR2 endocytosis: this figure is an extension of FIG. 17f , to show the results of additional treatment conditions, and for that reason, the top two and the bottom row images in this supplementary figure are identical to the images in FIG. 17 f:

FIG. 26a illustrates images of HUVEC that were preincubated with 50 μg/ml HDL₃, 0.1 μg/ml AIBP, or 50 μg/ml/ml HDL₃+0.1 μg/ml AIBP for 4 hours, then stimulated with 50 ng/ml VEGF for 20 min, fixed and stained with antibodies to VEGFR2 (red) and the early endosome marker EEA-1 (green); yellow arrows point to the surface localization of VEGFR2 and white arrows point to the endosomal localization of VEGFR2; scale, 10 μm;

FIG. 26b graphically illustrates the quantification of VEGFR2 and EEA-1 colocalization from FIG. 26a ; mean±SE (n=23 to 55); ****, p<0.0001; as further discussed in Example 3, below.

FIG. 27 (or Supplementary FIG. 10, Example 3): graphically illustrates the effect of human AIBP and HDL₃ on VEGF-induced signaling in HUVEC: this figure is the quantification of the signaling experiment shown in FIG. 17g ; normalized to β-tubulin and then to control (without VEGF); mean±SD (n=3); *, p<0.05; **, p<0.01; ***, p<0.001, as further discussed in Example 3, below.

FIG. 28 (or Supplementary FIG. 11, Example 3) in images and graphically illustrate that the addition of cholesterol reduces the effect of human AIBP and HDL₃ on VEGF-induced signaling in HUVEC:

FIG. 28a illustrates HUVEC preincubated with AIBP and HDL₃ as in FIG. 17g , followed by a one hour incubation with or without 20 μg/ml cholesterol-MβCD; the cells were then stimulated for 20 min with 50 ng/ml VEGF, and cell lysates were run on SDS-PAGE, blotted and probed as indicated;

FIG. 28b graphically illustrates the quantification of blot results; normalized to GAPDH and then to control, mean±SE (n=5); *, p<0.05;

as further discussed in Example 3, below.

FIG. 29 (or Supplementary FIG. 12, Example 3) in images and graphically illustrate the effect of human AIBP on VEGF-induced HUVEC migration:

FIG. 29A illustrates images of HUVEC preincubated with 50 μg/ml HDL₃, 0.1 μg/ml AIBP, or 50 μg/ml HDL₃+0.1 μg/ml AIBP for 4 hours, subjected to a migration assay as described in methods, Example 3, below; mean±SE (n=5). ***, p<0.001;

FIG. 29B graphically illustrates the data imaged in FIG. 29A,

as further discussed in Example 3, below.

FIG. 30 (or Supplementary FIG. 14, Example 3) illustrates images showing the expression of aibp2 mRNA in zebrafish embryos at 16 to 48 hpf; the images in this figure are to supplement FIG. 18a , which shows only a 24 hpf time point, as further discussed in Example 3, below.

FIG. 31 (or Supplementary FIG. 15, Example 3) illustrates images of Zebrafish Aibp binding to human ApoA-I:

FIG. 31a illustrates an SDS-PAGE and immunoblot where three pig of purified human ApoA-I were mixed with 3 μg of His-tagged zebrafish Aibp2 or human AIBP and then immunoprecipitated with an anti-His antibody; His-tagged ubiquitin was used as a negative control, the blots were probed as indicated;

FIG. 31b illustrates an SDS-PAGE and immunoblot showing Zebrafish Aibp binding to human HDL, where five μg of purified His-tagged DUB, Aibp1 or Aibp2 were incubated with 1 ml of human plasma overnight at 4° C.; ultracentrifugation was performed to separate lipoprotein fractions; and different fractions of lipoproteins were run on SDS-PAGE and immunoblotted with indicated antibodies;

FIG. 31c illustrates an agarose gel electrophoresis of human lipoprotein fractions separated by ultracentrifugation, stained with FatRed; DUB, deubiquitinase; Std, human standard plasma; LPDS, lipoprotein-deficient serum,

as further discussed in Example 3, below.

FIG. 32A and FIG. 32B (or Supplementary FIGS. 16 and 17, respectively, of Example 3):

FIG. 32A graphically illustrates the effect of zebrafish Aibp on cholesterol efflux from HUVEC to HDL₃: HUVEC were preloaded with ³H-cholesterol and then incubated for 1 hour with 50 μg/ml HDL₃ in the presence or absence of 0.2 μg/ml human AIBP, zebrafish Aibp1 or zebrafish Aibp2; the media and cell lysates were collected and ³H counts were measured; efflux was measured as the ³H counts in the media divided by the sum of ³H counts in the media and the cell lysates; mean±SD; n=3. **, p<0.01; *, p<0.05; #, p=0.08;

FIG. 32B graphically illustrates free cholesterol levels in aibp1 morphants: one-cell stage AB zebrafish embryos were injected with 8 ng of either control MO, aibp1 MO or aibp2 MO targeting ATG translation site; twenty four hpf embryos were fixed and stained with filipin to detect free cholesterol in embryos; intensities of filipin fluorescence in embryos (without yolks) were quantified. n=12-18. *, p<0.05;

as further discussed in Example 3, below.

FIG. 33 (or Supplementary FIG. 18, of Example 3) illustrates an SDS-PAGE and immunoblot showing the specificity of a zebrafish Aibp2 antibody: a polyclonal antibody against Aibp2 (guinea pig post-immune serum) was used to probe zebrafish homogenates; fifteen, 10 and 5 μl of the homogenates were loaded on an SDS gel, blotted and probed with the antibody, preincubated with 10 μg of either ubiquitine (Ub) or with Aibp2 (both recombinant proteins were produced from the same expression vector and under the same conditions); the blots were visualized with an anti-guinea pig IgG conjugated with HRP and a chemiluminescent substrate; as further discussed in Example 3, below.

FIG. 34A and FIG. 34B (or Supplementary FIGS. 19 and 20, respectively, of Example 3):

FIG. 34A graphically illustrates the quantification of the immunoblot experiment shown in FIG. 18f , showing the phosphorylation of signaling proteins in zebrafish lysates; Erk1 is the p44 MAPK; there were no statistically significant differences in the pErk2 (p42) values; normalized to β-tubulin and then to control; mean±SD (n=3 to 7). *, p<0.05; **, p<0.01; ***, p<0.001;

FIG. 34B illustrates low magnification, bright field images showing the area of fluorescent imaging of SeA (white box) and SIV (red box), which are displayed in FIG. 18g ; scale, 25 μm,

as further discussed in Example 3, below.

FIG. 35 (or Supplementary FIG. 21, of Example 3) illustrates dysregulated SeA and SIV angiogenesis in aibp2 morphants (splicing site):

FIG. 35a , FIG. 35b , and FIG. 35c illustrate where one-cell stage Tg(fli1:EGFP) zebrafish embryos were injected with 8 ng of either control MO or aibp2 MO2, targeting a splicing site; and the images of SeA (FIG. 35a , FIG. 35b ) and SIV (FIG. 35c ) were captured 3 days after MO injection; arrows point to dysregulated sprouts; scale, 50 μm;

FIG. 35d illustrate upper diagram schematically shows the targeted splicing site and gel (lower image) showing corresponding PCR products; PF and PR indicate the primers used for the amplification;

as further discussed in Example 3, below.

FIG. 36 (or Supplementary FIG. 22, of Example 3) illustrates the ectopic expression of Aibp2 inhibits SeA growth: one-cell stage Tg(fli1: egfp)^(y1) embryos were injected with 2 nl of 100 ng/μl DNA constructs for myog:Gal4, DsRed: 10×UAS:Aibp2 or myog: Gal4, DsRed: 10×UAS expression; images were captured at 30 hpf; arrows point to aberrant SeA growth, close to the sites of DsRed (and Aibp2) expression; scale, 25 μm; as further discussed in Example 3, below.

FIG. 37A and FIG. 37B (or Supplementary FIGS. 23 and 24, respectively, of Example 3):

FIG. 37A illustrates a WISH analysis of genes involved in angiogenesis: twenty four hpf control and aibp2 morphants were fixed and hybridized with probes to tie2 (GeneID:30747), vegfr2/kdrl (GeneID:796537), and fli1 (GeneID:30619); a 28 hpf time point was used for vegfr3/flt4 (GeneID:30121). cdh5 (Gene ID: 445471) at 24 hpf was used as an internal control; red arrows point to tie2 expression in the dorsal aorta, and orange brackets show the posterior cardinal vein; black arrows show increased expression of vegfr3 in the SeA of a aibp2 morphant;

FIG. 37B graphically illustrates the expression of genes involved in angiogenesis: total RNA was isolated from 24 hpf control and aibp2 morphants and qRT-PCR was performed with primers for tie2 (GeneID:30747), kdrb: (GeneID:554230), vegfr2/kdrl (GeneID:796537), and fli1 (GeneID:30619); the mRNA levels were normalized to β-actin (Gene ID: 57934) mRNA and then to the values in control embryos; the results are from a pool of 50 embryos per group, 3 to 4 replicates.***, p<0.001; **, p<0.01; *, p<0.05;

as further discussed in Example 3, below.

FIG. 38A and FIG. 38B (or Supplementary FIGS. 25 and 26, respectively, of Example 3):

FIG. 38A illustrates images showing the specificity of abcg1 and abca1a MOs:

FIG. 38A-a illustrates images of one-cell stage embryos injected with mRNA encoding mCherry-tagged N-terminus of Abcg1, with control or abcg1 MO; images were captured at 12 hpf;

FIG. 38A-b illustrates images showing one-cell stage embryos injected with mRNA encoding mCherry-tagged N-terminus of Abca1a, with control or abca1a MO. Images were captured at 4 hpf; scale, 100 μm;

FIG. 38B illustrates images and graphs showing free cholesterol levels in abca1a/abcg1 morphants:

FIG. 38B-a illustrates representative images of 24 hpf control and abca1a/abcg1 morphants stained with filipin to detect free cholesterol; note the yolks are artificially masked;

FIG. 38B-b graphically illustrates data showing the intensity of filipin fluorescence in images shown in panel FIG. 38B-a (without yolks) as quantified in 8 to 10 embryos per group; **, p<0.01;

as further discussed in Example 3, below.

FIG. 39A and FIG. 39B (or Supplementary FIGS. 27 and 28, respectively, of Example 3):

FIG. 39A graphically illustrates quantitative data for FIG. 19c ; the graphs show numbers of SeA ectopic branches and numbers and length of SIV ectopic sprouts from 5-12 embryos; ***, p<0.001;

FIG. 39A graphically illustrates quantification of the number of embryos with normal and abnormal angiogenesis (SeA with ectopic branching) resulting from injection of 8 ng control, abca1a or abcg1 MO, or 4 ng abca1a MO+4 ng abcg1 MO. 79-178 embryos per group; ***, p<0.001,

as further discussed in Example 3, below.

FIG. 40A and FIG. 40B (or FIGS. 1 and 2, respectively, of Example 4): FIG. 40A graphically illustrates tumor growth in mice as inhibited by injections of human AIBP; FIG. 40B graphically illustrates data showing that injections of AIBP inhibit proliferation of myeloid cells and boost proliferation of lymphocytes; as further discussed in Example 4, below.

FIG. 41 illustrates data showing that the conditional expression of Aibp in transgenic zebrafish abolishes high cholesterol diet (HCD)-induced vascular lipid accumulation:

FIG. 41A illustrates images of HCD-induced vascular lipid accumulation in control versus HCD conditions: red, fluorescent lipid tracer; green, GFP expressed in endothelial cells;

FIG. 41B schematically illustrates an exemplary DNA construct used to generate a transgenic zebrafish: gene expression is induced by heat shock, i.e., placing a zebrafish in a 37° C. water for 1 hour (normal maintenance temperature is 28° C.);

FIG. 41C graphically illustrates data showing that vascular lipid accumulation (as vascular lipid deposits as a percent of area) as induced in zebrafish by HCD feeding was abolished in animals with forced Aibp expression.

FIG. 42 graphically illustrates AIBP+HDL inhibit ROS generation and monocyte adhesion to endothelial cells:

FIG. 42A and FIG. 42 B: graphically illustrate data showing human aortic endothelial cells (HAEC) stimulated with LPS (FIG. 42A) or OxLDL (FIG. 42B), then treated with HDL, AIBP or their combination, and used in ROS generation (FIG. 42A) and THP-1 monocyte adhesion (FIG. 42B) assays;

FIG. 42C graphically illustrates data from ten plasma samples from patients with cardiovascular disease were tested in a monocyte adhesion assay; adding recombinant AIBP to plasma samples significantly reduced monocyte adhesion.

FIG. 43 graphically illustrates data showing that AIBP reduces lung inflammation in a mouse model of acute respiratory distress syndrome: all components were administered via nebulizer; neutrophils were counted in bronchoalveolar lavage fluid (BALF) 24 hors post LPS administration.

Like reference symbols in the various drawings indicate like elements.

Reference will now be made in detail to various exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. The following detailed description is provided to give the reader a better understanding of certain details of aspects and embodiments of the invention, and should not be interpreted as a limitation on the scope of the invention.

DETAILED DESCRIPTION

In alternative embodiments, the invention provides pharmaceutical compounds and formulations comprising nucleic acids and polypeptides for regulating or manipulating (including enhancing expression, upregulating or inhibiting) the expression of ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP), and methods for making and using them.

Cholesterol is essential for cellular function and its levels are tightly regulated by several mechanisms, including cholesterol efflux to HDL and apoA-I. We found that proper removal of cholesterol from endothelial cells (EC) is required for normal embryonic angiogenesis and that apoA-I binding protein (AIBP) plays a critical role in this process. AIBP knockdown resulted in dysregulated sprouting angiogenesis, while ectopic AIBP expression inhibited angiogenesis during zebrafish development. A non-cell autonomous character of the AIBP effect suggested its role similar to that of a repellent molecule guiding angiogenesis. Importantly, dysregulated angiogenesis was phenocopied in embryos deficient in cholesterol transporters ABCA1 and ABCG1. Cholesterol levels were increased in AIBP- and ABCA1/G1-deficient embryos. Our findings demonstrate that AIBP accelerates cholesterol efflux from EC and that this mechanism is involved in regulation of angiogenesis.

The invention also demonstrates that ApoA-I binding protein (AIBP) accelerates ABCG1-dependent cholesterol efflux from endothelial cells (EC) and that AIBP expression is critical for proper angiogenesis. Thus, in alternative embodiments, the invention provides compositions and methods using ApoA-I binding protein (AIBP) to accelerate ABCG1-dependent cholesterol efflux from endothelial cells (EC) and to control AIBP expression and regulate or modify angiogenesis.

In the presence of HDL, AIBP reduces lipid rafts and inhibits vascular endothelial growth factor (VEGF)-induced EC tube formation in vitro and aortic ring neovascularization ex vivo. Thus, in alternative embodiments, the invention provides compositions and methods using ApoA-I binding protein (AIBP) to reduce lipid rafts and inhibits vascular endothelial growth factor (VEGF)-induced EC tube formation in vivo and in vitro.

The invention demonstrates that AIBP functions as a non-cell autonomous regulator of angiogenesis in embryonic zebrafish. AIBP knockdown results in dysregulated sprouting/branching angiogenesis, while ectopic Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1/Abcg1-deficient embryos, and cholesterol levels are increased in Aibp-deficient and Abca1/Abcg1-deficient embryos. Our findings demonstrate that secreted AIBP positively regulates cholesterol efflux from EC and that effective cholesterol efflux is critical for proper angiogenesis.

Products of Manufacture, Kits

The invention also provides products of manufacture, kits and pharmaceuticals for practicing the methods of this invention. In alternative embodiments, the invention provides products of manufacture, kits and/or pharmaceuticals comprising all the components needed to practice a method of the invention.

Antisense Inhibitory Nucleic Acid Molecules

In alternative embodiments, the invention provides compositions and methods for reducing the amount of or reducing the activity of APOA1BP by administering APOA1BP-inhibiting nucleic acids, e.g., an antisense morpholino oligonucleotide (MO), an miRNA, an siRNA and the like. In alternative embodiments, APOA1BP-inhibiting pharmaceutical compositions and formulations of the invention are administered to an individual in need thereof in an amount sufficient to stimulate tissue revascularization, e.g., supporting or stimulating revascularization of heart tissue, e.g., after a cardiac ischemia.

In alternative embodiments, compositions and methods of the invention comprise use of an inhibitory nucleic acid molecule or an antisense oligonucleotide inhibitory to expression of an APOA1BP gene transcript. In alternative embodiments, compositions and methods of the invention comprise use of an inhibitory nucleic acid molecule or antisense oligonucleotide inhibitory to expression of the APOA1BP gene, e.g., an APOA1BP1 or APOA1BP2 gene transcript comprises: an RNAi inhibitory nucleic acid molecule, a double-stranded RNA (dsRNA) molecule, a small interfering RNA (siRNA), a microRNA (miRNA) and/or a short hairpin RNA (shRNA), or a ribozyme.

Naturally occurring or synthetic nucleic acids can be used as antisense oligonucleotides. The antisense oligonucleotides can be of any length; for example, in alternative aspects, the antisense oligonucleotides are between about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40. The optimal length can be determined by routine screening. The antisense oligonucleotides can be present at any concentration. The optimal concentration can be determined by routine screening. A wide variety of synthetic, non-naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem. For example, peptide nucleic acids (PNAs) containing non-ionic backbones, such as N-(2-aminoethyl) glycine units can be used. Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N. J., 1996). Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholino carbamate nucleic acids.

RNA Interference (RNAi)

In one aspect, the invention provides RNAi inhibitory nucleic acid molecules capable of decreasing or inhibiting expression of one or a set of APOA1BP transcripts or proteins, e.g., the transcript (mRNA, message) or isoform or isoforms thereof. In one aspect, the RNAi molecule comprises a double-stranded RNA (dsRNA) molecule. The RNAi molecule can comprise a double-stranded RNA (dsRNA) molecule, e.g., siRNA, miRNA (microRNA) and/or short hairpin RNA (shRNA) molecules.

In alternative aspects, the RNAi is about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length. While the invention is not limited by any particular mechanism of action, the RNAi can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs. When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). A possible basic mechanism behind RNAi, e.g., siRNA for inhibiting transcription and/or miRNA to inhibit translation, is the breaking of a double-stranded RNA (dsRNA) matching a specific gene sequence into short pieces called short interfering RNA, which trigger the degradation of mRNA that matches its sequence.

In one aspect, intracellular introduction of the RNAi (e.g., miRNA or siRNA) is by internalization of a target cell specific ligand bonded to an RNA binding protein comprising an RNAi (e.g., microRNA) is adsorbed. The ligand can be specific to a unique target cell surface antigen. The ligand can be spontaneously internalized after binding to the cell surface antigen. If the unique cell surface antigen is not naturally internalized after binding to its ligand, internalization can be promoted by the incorporation of an arginine-rich peptide, or other membrane permeable peptide, into the structure of the ligand or RNA binding protein or attachment of such a peptide to the ligand or RNA binding protein. See, e.g., U.S. Patent App. Pub. Nos. 20060030003; 20060025361; 20060019286; 20060019258. In one aspect, the invention provides lipid-based formulations for delivering, e.g., introducing nucleic acids of the invention as nucleic acid-lipid particles comprising an RNAi molecule to a cell, see e.g., U.S. Patent App. Pub. No. 20060008910.

Methods for making and using RNAi molecules, e.g., siRNA and/or miRNA, for selectively degrade RNA are well known in the art, see, e.g., U.S. Pat. Nos. 6,506,559; 6,511,824; 6,515,109; 6,489,127.

Methods for making expression constructs, e.g., vectors or plasmids, from which an inhibitory polynucleotide (e.g., a duplex siRNA of the invention) is transcribed are well known and routine. A regulatory region (e.g., promoter, enhancer, silencer, splice donor, acceptor, etc.) can be used to transcribe an RNA strand or RNA strands of an inhibitory polynucleotide from an expression construct. When making a duplex siRNA inhibitory molecule, the sense and antisense strands of the targeted portion of the targeted IRES can be transcribed as two separate RNA strands that will anneal together, or as a single RNA strand that will form a hairpin loop and anneal with itself. For example, a construct targeting a portion of a gene, e.g., a APOA1BP coding sequence or transcriptional activation sequence, is inserted between two promoters (e.g., mammalian, viral, human, tissue specific, constitutive or other type of promoter) such that transcription occurs bidirectionally and will result in complementary RNA strands that may subsequently anneal to form an inhibitory siRNA of the invention.

Alternatively, a targeted portion of a gene, coding sequence, promoter or transcript can be designed as a first and second antisense binding region together on a single expression vector; for example, comprising a first coding region of a targeted gene in sense orientation relative to its controlling promoter, and wherein the second coding region of the gene is in antisense orientation relative to its controlling promoter. If transcription of the sense and antisense coding regions of the targeted portion of the targeted gene occurs from two separate promoters, the result may be two separate RNA strands that may subsequently anneal to form a gene-inhibitory siRNA used to practice this invention.

In another aspect, transcription of the sense and antisense targeted portion of the targeted gene is controlled by a single promoter, and the resulting transcript will be a single hairpin RNA strand that is self-complementary, i.e., forms a duplex by folding back on itself to create a gene-inhibitory siRNA molecule. In this configuration, a spacer, e.g., of nucleotides, between the sense and antisense coding regions of the targeted portion of the targeted gene can improve the ability of the single strand RNA to form a hairpin loop, wherein the hairpin loop comprises the spacer. In one embodiment, the spacer comprises a length of nucleotides of between about 5 to 50 nucleotides. In one aspect, the sense and antisense coding regions of the siRNA can each be on a separate expression vector and under the control of its own promoter.

Inhibitory Ribozymes

In alternative embodiment, compositions and methods of the invention comprise use of ribozymes capable of binding and inhibiting, e.g., decreasing or inhibiting, expression of one or a set of APOA1BP transcripts or proteins, or isoform or isoforms thereof

These ribozymes can inhibit a gene's activity by, e.g., targeting a genomic DNA or an mRNA (a message, a transcript). Strategies for designing ribozymes and selecting a gene-specific antisense sequence for targeting are well described in the scientific and patent literature, and the skilled artisan can design such ribozymes using the novel reagents of the invention. Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA. Thus, the ribozyme recognizes and binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence. After a ribozyme has bound and cleaved its RNA target, it can be released from that RNA to bind and cleave new targets repeatedly.

Formulations and Pharmaceutical Compositions

In alternative embodiments, the invention provides pharmaceutical formulations or compositions comprising nucleic acids and polypeptides for regulating (including upregulating or inhibiting) the expression of ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP). In alternative embodiments, the invention provides pharmaceutical formulations or compositions for use in in vivo, in vitro or ex vivo methods to treat, prevent, reverse and/or ameliorate neovascularization of a growth, cancer or tumor whose growth or survival depends on neovascularization. In alternative embodiments, pharmaceutical compositions and formulations of the invention that comprise APOA1BP nucleic acids and polypeptides or result in an increase in expression or activity of APOA1BP nucleic acids and polypeptides are administered to an individual in need thereof in an amount sufficient to treat, prevent, reverse and/or ameliorate a dyslipidemia, e.g., to treat, prevent, reverse and/or ameliorate conditions responsive to increasing cholesterol efflux from cells, including cardiovascular disease and atherosclerosis. In alternative embodiments, pharmaceutical compositions and formulations of the invention that comprise APOA1BP nucleic acids and polypeptides or result in an increase in expression or activity of APOA1BP nucleic acids and polypeptides are administered to an individual in need thereof in an amount sufficient to stimulate tissue revascularization, e.g., supporting or stimulating revascularization of heart tissue, e.g., after a cardiac ischemia.

In alternative embodiments, the pharmaceutical compositions of the invention can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration are well described in the scientific and patent literature, see, e.g., the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing Co., Easton Pa. (“Remington's”). For example, in alternative embodiments, these compositions of the invention are formulated in a buffer, in a saline solution, in a powder, an emulsion, in a vesicle, in a liposome, in a nanoparticle, in a nanolipoparticle and the like. In alternative embodiments, the compositions can be formulated in any way and can be applied in a variety of concentrations and forms depending on the desired in vivo, in vitro or ex vivo conditions, a desired in vivo, in vitro or ex vivo method of administration and the like. Details on techniques for in vivo, in vitro or ex vivo formulations and administrations are well described in the scientific and patent literature. Formulations and/or carriers used to practice this invention can be in forms such as tablets, pills, powders, capsules, liquids, gels, syrups, slurries, suspensions, etc., suitable for in vivo, in vitro or ex vivo applications.

In practicing this invention, the compounds (e.g., formulations) of the invention can comprise a solution of compositions (which include peptidomimetics, racemic mixtures or racemates, isomers, stereoisomers, derivatives and/or analogs of compounds of the invention) disposed in or dissolved in a pharmaceutically acceptable carrier, e.g., acceptable vehicles and solvents that can be employed include water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any fixed oil can be employed including synthetic mono- or diglycerides, or fatty acids such as oleic acid. In one embodiment, solutions and formulations used to practice the invention are sterile and can be manufactured to be generally free of undesirable matter. In one embodiment, these solutions and formulations are sterilized by conventional, well known sterilization techniques.

The solutions and formulations used to practice the invention can comprise auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and can be selected primarily based on fluid volumes, viscosities and the like, in accordance with the particular mode of in vivo, in vitro or ex vivo administration selected and the desired results.

The compositions and formulations of the invention can be delivered by the use of liposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells (e.g., an injured or diseased neuronal cell or CNS tissue), or are otherwise preferentially directed to a specific tissue or organ type, one can focus the delivery of the active agent into a target cells in an in vivo, in vitro or ex vivo application.

Nanoparticles, Nanolipoparticles and Liposomes

The invention also provides nanoparticles, nanolipoparticles, vesicles and liposomal membranes comprising compounds used to practice the methods of this invention, e.g., to deliver compositions of the invention (which include APOA1BP nucleic acids and polypeptides of the invention) to mammalian cells in vivo, in vitro or ex vivo. In alternative embodiments, these compositions are designed to target specific molecules, including biologic molecules, such as polypeptides, including cell surface polypeptides, e.g., for targeting a desired cell type, e.g., a myocyte or heart cell, and endothelial cell, and the like.

The invention provides multilayered liposomes comprising compounds used to practice this invention, e.g., as described in Park, et al., U.S. Pat. Pub. No. 20070082042. The multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition used to practice this invention.

Liposomes can be made using any method, e.g., as described in Park, et al., U.S. Pat. Pub. No. 20070042031, including method of producing a liposome by encapsulating an active agent (e.g., APOA1BP nucleic acids and polypeptides), the method comprising providing an aqueous solution in a first reservoir; providing an organic lipid solution in a second reservoir, and then mixing the aqueous solution with the organic lipid solution in a first mixing region to produce a liposome solution, where the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce a liposome encapsulating the active agent; and immediately then mixing the liposome solution with a buffer solution to produce a diluted liposome solution.

In one embodiment, liposome compositions used to practice this invention comprise a substituted ammonium and/or polyanions, e.g., for targeting delivery of a compound (e.g., a APOA1BP nucleic acid and polypeptide of the invention) used to practice this invention to a desired cell type (e.g., an endothelial cell, a cancer cell, or any tissue in need thereof), as described e.g., in U.S. Pat. Pub. No. 20070110798.

The invention also provides nanoparticles comprising compounds (e.g., APOA1BP nucleic acids and polypeptides of the invention) used to practice this invention in the form of active agent-containing nanoparticles (e.g., a secondary nanoparticle), as described, e.g., in U.S. Pat. Pub. No. 20070077286. In one embodiment, the invention provides nanoparticles comprising a fat-soluble active agent of this invention or a fat-solubilized water-soluble active agent to act with a bivalent or trivalent metal salt.

In one embodiment, solid lipid suspensions can be used to formulate and to deliver compositions used to practice this invention to mammalian cells in vivo, in vitro or ex vivo, as described, e.g., in U.S. Pat. Pub. No. 20050136121.

Delivery Vehicles

In alternative embodiments, any delivery vehicle can be used to practice the methods or used to practice this invention, e.g., to deliver compositions of the invention (e.g., APOA1BP nucleic acids and polypeptides) to mammalian cells in vivo, in vitro or ex vivo. For example, delivery vehicles comprising polycations, cationic polymers and/or cationic peptides, such as polyethyleneimine derivatives, can be used e.g. as described, e.g., in U.S. Pat. Pub. No. 20060083737.

In one embodiment, a dried polypeptide-surfactant complex is used to formulate a composition used to practice this invention, e.g. as described, e.g., in U.S. Pat. Pub. No. 20040151766.

In one embodiment, a composition used to practice this invention can be applied to cells using vehicles with cell membrane-permeant peptide conjugates, e.g., as described in U.S. Pat. Nos. 7,306,783; 6,589,503. In one aspect, the composition to be delivered is conjugated to a cell membrane-permeant peptide. In one embodiment, the composition to be delivered and/or the delivery vehicle are conjugated to a transport-mediating peptide, e.g., as described in U.S. Pat. No. 5,846,743, describing transport-mediating peptides that are highly basic and bind to poly-phosphoinositides.

In one embodiment, electro-permeabilization is used as a primary or adjunctive means to deliver the composition to a cell, e.g., using any electroporation system as described e.g. in U.S. Pat. Nos. 7,109,034; 6,261,815; 5,874,268.

Dosaging

The pharmaceutical compositions and formulations of the invention can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a subject already suffering from a disease, condition, infection or defect in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disease, condition, infection or disease and its complications (a “therapeutically effective amount”). For example, in alternative embodiments, APOA1BP nucleic acid- or polypeptide-comprising pharmaceutical compositions and formulations of the invention are administered to an individual in need thereof in an amount sufficient to treat, prevent, reverse and/or ameliorate a dyslipidemia, unwanted angiogenesis, e.g., cancer or tumor angiogenesis, or to stimulate wanted angiogenesis, e.g., to stimulate revascularization of heart tissue, e.g., after a cardiac ischemia.

The amount of pharmaceutical composition adequate to accomplish this is defined as a “therapeutically effective dose.” The dosage schedule and amounts effective for this use, i.e., the “dosing regimen,” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.

The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84:1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24:103-108; the latest Remington's, supra). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods of the invention are correct and appropriate.

Single or multiple administrations of formulations can be given depending on the dosage and frequency as required and tolerated by the patient. The formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate a conditions, diseases or symptoms as described herein. For example, alternative exemplary pharmaceutical formulations for oral administration of compositions used to practice the invention are in a daily amount of between about 0.1 to 0.5 to about 20, 50, 100 or 1000 or more ug per kilogram of body weight per day. In an alternative embodiment, dosages are from about 1 mg to about 4 mg per kg of body weight per patient per day are used. Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ. Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation. Actual methods for preparing parenterally or non-parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's, supra.

The methods of the invention can further comprise co-administration with other drugs or pharmaceuticals, e.g., compositions for treating any neurological or neuromuscular disease, condition, infection or injury, including related inflammatory and autoimmune diseases and conditions, and the like. For example, the methods and/or compositions and formulations of the invention can be co-formulated with and/or co-administered with, fluids, antibiotics, cytokines, immunoregulatory agents, anti-inflammatory agents, pain alleviating compounds, complement activating agents, such as peptides or proteins comprising collagen-like domains or fibrinogen-like domains (e.g., a ficolin), carbohydrate-binding domains, and the like and combinations thereof.

Bioisosteres of Compounds of the Invention

In alternative embodiment, the invention also provides and uses bioisosteres of compounds of the invention, e.g., polypeptides having a APOA1BP activity. Bioisosteres of the invention are compounds of the invention, e.g., APOA1BP nucleic acids and polypeptides, comprising one or more substituent and/or group replacements with a substituent and/or group having substantially similar physical or chemical properties which produce substantially similar biological properties to compounds of the invention. In one embodiment, the purpose of exchanging one bioisostere for another is to enhance the desired biological or physical properties of a compound without making significant changes in chemical structures.

For example, in one embodiment, one or more hydrogen atom(s) is replaced with one or more fluorine atom(s), e.g., at a site of metabolic oxidation; this may prevent metabolism (catabolism) from taking place. Because the fluorine atom is similar in size to the hydrogen atom the overall topology of the molecule is not significantly affected, leaving the desired biological activity unaffected. However, with a blocked pathway for metabolism, the molecule may have a longer half-life or be less toxic, and the like.

Kits and Instructions

The invention provides kits comprising compositions and/or instructions for practicing methods of the invention. As such, kits, cells, vectors and the like can also be provided. In alternative embodiments, the invention provides kits comprising: a composition used to practice a method of any of the invention, or a composition, a pharmaceutical composition or a formulation of the invention, and optionally comprising instructions for use thereof.

The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.

EXAMPLES Example 1 Exemplary Compounds and Methods of the Invention Regulate Cholesterol Transport and Angiogenesis: ApoA-I Binding Protein-Mediated Cholesterol Efflux Controls Angiogenesis

This example describes and demonstrates exemplary activities, and the efficacy, of compounds and methods of the invention. While the invention is not limited by any particular mechanism of action, in one embodiment, methods of the invention use APOA1BP to accelerate cholesterol efflux from cells, e.g., endothelial cells (EC), and that this mechanism is involved in regulation of angiogenesis, and secreted AIBP functions as a local regulator of reverse cholesterol transport and as such, controls endothelial cell (EC) proliferation and angiogenesis.

AIBP is differentially expressed in many angiogenic tumors, making to a top 1% of differentially regulated genes in invasive breast carcinoma (11, 12). Thus, in this work, we tested the hypothesis that secreted AIBP functions as a local regulator of reverse cholesterol transport and as such, controls endothelial cell (EC) proliferation and angiogenesis.

AIBP is evolutionary conserved from Drosophila to zebrafish to mouse and human (FIG. S1A). Zebrafish AIBP (gene name apoalbp) has two isoforms, AIBP1 and AIBP2 (FIG. SIB). To identify the role that AIBP plays in embryonic angiogenesis, we injected antisense morpholino oligonucleotides (MO) targeting AIBP1 and AIBP2 translation sites into one-cell stage embryos of Tg(fli1-egfp)^(y1) zebrafish, which express EGFP in EC (13). No obvious abnormalities were observed in AIBP1 morphants (data not shown), whereas ectopic branching of segmental arteries (SeA) and pathologic sprouting of subintestinal veins (SW) were found in AIBP2 morphants (FIG. 1A-B).

These results were confirmed with a morpholino oligonucleotide (MO) targeting AIBP2 gene splicing (FIG. S2). The AIBP2 knockdown in morphant embryos was validated in western blot (FIG. 2C). The unregulated sprouting of SeAs in AIBP2 morphants was partially rescued by ectopic expression of AIBP2 delivered by an mRNA lacking the binding site targeted by the MO (FIG. 1B). The results of these loss of function and rescue experiments demonstrated a role for AIBP2 in angiogenesis.

To test whether AIBP2 deficiency affected EC proliferation, we injected control and AIBP2 MO into Tg(fli1:negfp)^(y7) one-cell stage embryos. Numbers of fluorescent EC nuclei in SeAs and DLAV of AIBP2 morphants were increased (FIG. 1C-D), suggesting that AIBP2 regulates EC proliferation.

Next, the tissue distribution of AIBP2 in embryos at different developmental stages was examined by whole embryo in situ hybridization (WISH) (FIG. 1E). AIBP2 expression was detected as early as 20 min post fertilization and was clearly evident in somites at 12 hours post fertilization (hpf). Notably, the AIBP2 signal at 24 hpf showed an interval somite distribution in the medial and posterior trunk of the embryo, the distribution resembling that of type 3 semaphorins, non-cell autonomous repellent cues that guide the patterning of developing SeAs via PlexinD1 receptors (14-16).

To determine the cell autonomy of AIBP2 effect on angiogenesis, we performed cell transplantation experiments. Fluorescent EC from wild type Tg(fli1:egfp)^(y1) donors found in nonfluorescent AIBP2 morphants displayed excessive sprouting, branching and filopodial projections (FIG. 1F-G).

In a reverse experiment, transplantation of cells from Tg(fli1:egfp)^(y1) AIBP2 morphants into wild type embryos with unaffected AIBP2 expression did not disturb normal morphology of fluorescent EC. These results demonstrate the role of AIBP2 as a repellent molecule whose function depends on the milieu surrounding EC but not on AIBP2 expression in the EC themselves.

Notch, VEGF and other signaling pathways are finely tuned during the process of angiogenesis (17-21). The impact of the loss of AIBP2 on molecular pathways involved in angiogenesis was examined in control and AIBP2 morphants in a microarray experiment. Genes indicative of enhanced cell proliferation and genes involved in angiogenesis, such as Tie2, VEGFR2, VEGFR3 and fli1, were upregulated in AIBP2 morphants (FIG. S3). The increased expression of genes involved in angiogenesis was confirmed by both qRT-PCR and WISH (FIGS. 2A and 2B). Further, in agreement with the excessive SeA branching/sprouting phenotype (FIGS. 1A and D), Dll4 was markedly down-regulated in AIBP2 morphants (FIG. 2B). Taken together, these data demonstrate that the molecular pathways that orchestrate angiogenesis are dramatically affected by the AIBP2 knockdown.

The interplay between activation and inhibition of ERK and Akt directs angiogenesis, and upregulated Akt activity rescues the angiogenesis defects caused by VEGFR inhibitors (22-24). Lysates of AIBP2 morphant embryos displayed robust activation of Akt and mild inhibition of ERK (FIG. 2C), supporting the AIBP2 role in angiogenesis. The AIBP2 knockdown in morphants was confirmed with a guinea pig antibody generated against recombinant zebrafish AIBP2 (FIGS. 2C and S4).

Because binding of human AIBP to apoA-I (8) suggests a role for AIBP in cholesterol metabolism, we tested whether AIBP2 knockdown affects cholesterol levels in embryonic zebrafish. The 24 hpf embryos were stained with filipin, a fluorescent dye specifically binding to free (unesterified) cholesterol (25). The filipin fluorescence was increased in AIBP2 morphant embryos (excluding the yolk) compared to controls (FIGS. 2D and E). These results were confirmed with direct gas chromatography measurements of free cholesterol in lipid extracts from pooled embryos from which the yolk was removed (FIG. 3F). Increased levels of free cholesterol measured by two independent techniques suggest that AIBP2 is required for normal cholesterol metabolism.

Next, we sought to connect a putative role of AIBP in cholesterol metabolism with its role in angiogenesis discovered in this study. First, we tested if zebrafish AIBP2 binds with HDL and apoA-I. AIBP2 was added to human plasma and lipoprotein fractions were separated by density ultracentrifugation (FIG. S5). AIBP2 was detected in the HDL fraction but not in LDL or VLDL fractions (FIG. 3A). A pull down assay was used to confirm binding of human AIBP and zebrafish AIBP2 with human apoA-I (FIG. 3B). ApoA-I and HDL remove cholesterol from the cells.

We asked whether AIBP binding to HDL and apoA-I would have any effect on cholesterol efflux from EC. Within 1 hour, human AIBP and zebrafish AIBP2 significantly increased cholesterol efflux to HDL from human umbilical vein endothelial cells (HUVEC), while zebrafish AIBP1 had a smaller effect (FIG. 3C). Accelerated cholesterol efflux in the presence of human AIBP was observed within first 4 hours (FIG. 3D), and the enhancement of cholesterol removal in the presence of AIBP was diminished at later time points. These results demonstrate that AIBP promotes cholesterol efflux from EC.

Because cholesterol levels in the plasma membrane microdomains may drastically affect cell signaling (26, 27), we tested whether enhanced cholesterol efflux in cells preincubated with hAIBP/HDL would affect subsequent HUVEC responses to VEGF. The VEGF-stimulated activation of Akt was decreased in HUVEC preincubated with hAIBP/HDL, but not with AIBP or HDL alone (FIG. S6). Likewise, hAIBP/HDL completely abrogated VEGF-induced HUVEC angiogenesis in an in vitro assay (FIG. 3E). These HUVEC results agree with the increased Akt phosphorylation (FIG. 2C) and increased EC branching (FIG. 1A) observed in AIBP2 zebrafish morphants.

In HUVEC, ATP-binding cassette sub-family G member 1 (ABCG1) is a major transporter responsible for cholesterol efflux (28). Remarkably, knockdown of ABCG1 in HUVEC resulted in complete loss of the AIBP effect (FIG. 3F), suggesting that AIBP indeed accelerates cholesterol efflux from HUVEC via the HDL/ABCG1 pathway.

To further test the hypothesis that effective cholesterol efflux is required for normal angiogenesis, we knocked down zebrafish ABCA1 and ABCG1. These cellular cholesterol transporters are essential for cholesterol efflux in mammals (1-3). Expression of ABCA1 and ABCG1 has been found in zebrafish and, as in mammals, it is induced by liver X receptor agonists (29, 30). We found that the loss of ABCA1 and ABCG1 in zebrafish closely reproduced the vascular developmental defects of AIBP2 morphants, in both SeA and, markedly, in SIV vascular beds (FIG. 4A-F). The latter finding was confirmed with Tg(fli1:negfp)y7 AIBP2 and ABCA1/G1 morphants in which we observed increased numbers of EC nuclei in SIV sprouts (FIG. S7). As expected, ABCA1/G1 morphants had higher levels of free cholesterol than control embryos (FIG. S8). Similarly to AIBP2 morphants, phosphorylation of Akt was increased in ABCA1/G1 morphants as well (FIG. 2C).

In agreement with the AIBP2 knockdown results, overexpression of AIBP2-mCherry in somites driven by the myog promoter resulted in inhibition of SeA growth and incomplete SeA angiogenesis (FIG. 4G-H). Remarkably, the inhibition of SeA formation induced by ectopic AIBP2-mCherry expression was rescued by knocking down ABCA1/G1. These results provide an additional evidence that AIBP2 is a repellent guiding blood vessel growth and also suggest that ABCA1/G1 are required for AIBP2 function in reverse cholesterol transport. The latter is supported by the results of HUVEC experiments (FIG. 3F).

Our findings demonstrating the role of ABCA1/G1 in regulation of EC branching/sprouting in zebrafish embryo agree with the recent reports suggesting the role of reverse cholesterol transport in proliferation of other cell types in adult organisms (4-6, 31, 32). In mice, deficiency in ABCA1 and/or ABCG1 results in elevated cellular cholesterol levels and enhanced proliferation of monocytes, smooth muscle cells and T cells as well as dysregulated cell migration (4-6, 31, 32). Importantly, crossing ABCA1/G1 double knockout mice with ApoA-I transgenic mice rescued the myeloproliferative disorder of monocytes in ABCA1/G1-deficient mice, indicating that HDL inhibits abnormal monocyte proliferation by reducing cholesterol content in the cells (4).

High levels of cholesterol in apoE null mice have been reported to facilitate endothelial progenitor cell-mediated repair after incurred carotid artery injury (31). Cholesterol has been shown to play a critical role in lipid rafts formation (33). Increased signaling of IL3fA receptor in myeloid cells from ABCA1/G1 double knockout mice and enhanced TLR4 signaling in macrophages from ABCA1 knockout mice are likely the result of cholesterol-induced enrichment in lipid rafts leading to receptor dimerization (4, 34). In agreement, cholesterol depletion inhibits T cell activation (33).

Our results demonstrate, for the first time, the importance of cholesterol efflux in regulation of embryonic angiogenesis. In addition, a novel finding of this study is that AIBP plays a previously unrecognized role in accelerated cholesterol efflux from EC and in angiogenesis. Increased cholesterol efflux, promoted by AIBP, likely disrupts organization of plasma membrane microdomains and thereby compromises function of membrane receptors, such as VEGFR and Tie2. This may explain the connection between intracellular levels of free cholesterol and EC branching/sprouting. As illustrated in FIG. 4I, our findings imply that in embryonic zebrafish angiogenesis, AIBP2 expression in somites likely recruits HDL to sites around growing SeA.

Increased cholesterol efflux at these sites, together with other inhibitory mechanisms (14, 15), arrests aberrant intrasomitic growth of EC, thereby guiding angiogenesis along the path between somites. The invention provides compositions and methods to manipulate AIBP function in angiogenesis and in a variety of pathologic and regenerative processes, from cancer to cardiovascular disease.

References Example 1

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FIGURE LEGENDS FIG. 1. Role of AIBP in Zebrafish Angiogenesis

-   -   (A) One-cell stage fli1:EGFP zebrafish embryos were injected         with 8 ng of either control MO or AIBP2 MO targeting ATG         translation site. The images of SeA (images 1-2) and of SIV         (3-4) were captured 3 days after MO injection. Arrows point to         dysregulated sprouts. Scale, 50 m.     -   (B) Numbers of embryos with abnormal angiogenesis resulting from         injection of control or AIBP2 MO. The abnormal angiogenesis was         partially rescued by co-injection of 40 pg of AIBP2 RNA lacking         the MO targeting site. 100-149 embryos per group. ***, p<0.001.     -   (C) One-cell stage fli1:nEGFP embryos were injected with 8 ng of         either control MO or AIBP2 MO, and images were captured at 3         dpf. EC nuclei in each SeA were numerated. Scale, 50 μm.     -   (D) Quantification of EC nuclei in SeAs, n=8-9.     -   (E) Tissue distribution of AIBP2 mRNA in zebrafish embryo.         Embryos at different developmental stages were fixed and WISH         was performed with an antisense AIBP2 probe. Scale, 100 μm.     -   (F) Mosaic expression analysis of EC branching in WT and AIBP2         knockdown (KD) embryos. Four hours post fertilization; cells         were isolated from donor embryos and were transplanted into         recipient embryos (details of donor and recipient embryos are         depicted above images). Three days after transplantation, the         recipient embryos were analyzed. Red arrowheads indicate         aberrant ectopic branches/sprouts in the AIBP2 KD recipients.         Scale, 20 μm.     -   (G) Numbers of ectopic branches/filopodial projections per SeA.         8-16 embryos per group. ***, p<0.001.

FIG. 2. Upregulated Markers of EC Proliferation and Angiogenesis in AIBP2 Morphants

-   -   (A) Expression of genes involved in angiogenesis. Total RNA was         isolated from 24 hpf control and AIBP2 morphants and qRT-PCR was         performed. The results are normalized to actin. The results are         of 50 embryos per group, 3 to 4 replicates.***, p<0.001; **,         p<0.01; *, p<0.05.     -   (B) WISH analysis of genes involved in angiogenesis. Twenty four         hpf control and AIBP2 morphants were fixed and hybridized with         indicated probes. Scale, 100 μm.     -   (C) Akt and ERK1/2 phosphorylation. Twenty four hpf control and         AIBP2 morphants were lysed and the lysates separated on SDS-PAGE         and immunoblotted as indicated.     -   (D-F) Free cholesterol levels in AIBP2 morphants. (D) Twenty         four hpf control and AIBP2 morphants were fixed and stained with         filipin to detect free cholesterol in embryos. Note the yolks         are artificially masked on the images. (E) Intensities of         filipin fluorescence in mages shown in panel F (without yolks)         were quantified in 12-15 embryos per group. *, p<0.05.     -   (F) One-cell stage embryos were injected with control or AIBP2         MO. At 24 hpf, the trunk area (without yolk and head) was         dissected and total lipid was extracted. Free cholesterol levels         were determined by GC. The cholesterol levels were normalized to         the protein content and then to the values in control MO         embryos. 50-70 embryos were pooled for each sample. The data are         from 3-4 independent experiments.

FIG. 3. Effect of AIBP on Cholesterol Efflux

-   -   (A) Five μg of purified His-tagged deubiquitinase (DUB, a         non-relevant protein of the same molecular weight as AIBP),         AIBP1 or AIBP2 were incubated with 1 ml of human plasma         overnight at 4° C. Ultracentrifugation was performed to separate         lipoprotein fractions. Different fractions of lipoproteins were         run on SDS-PAGE and immunoblotted with indicated antibodies.     -   (B) Three μg of purified human ApoA-I were mixed with 3 μg         His-tagged AIBP2 or hAIBP and then immunoprecipitated with an         á-His antibody. The blots were probed as indicated.     -   (C) Cholesterol efflux to HDL3. HUVEC were preloaded with         3H-cholesterol and then incubated for 1 hour with media, 50         μg/ml HDL3, 50 μg/ml HDL3+0.2 μg/ml His-AIBP, or 50 μg/ml         HDL3+0.2 μg/ml His-DUB. The media and cell lysates were         collected and 3H counts were measured. Efflux was measured as         the 3H counts in the medium divided by the sum of 3H counts in         the medium and the cell lysates. Mean±SD; n=3. **, p<0.01; *,         p<0.05; #, p=0.08.     -   (D) Kinetics of cholesterol efflux from HUVEC to HDL3.         Experimental conditions as in C.     -   (E) Effect of hAIBP and HDL3 on EC tube formation. HUVEC were         preincubated with 50 μg/ml HDL3, 0.1 μg/ml His-hAIBP, or 50         μg/ml/ml HDL3+0.1 μg/ml His-hAIBP for 4 hours. Cells were then         seeded on MATRIGEL™, in the presence or absence of 20 ng/ml         VEGF, and imaged following a 12 hour incubation. Scale, 100 μm.     -   (F) Effect of ABCG1 knockdown on hAIBP mediated-cholesterol         efflux. HUVEC were transfected with control or ABCG1 siRNA. The         ABCG1 knockdown was confirmed in western blot. Control and ABCG1         deficient HUVEC were used in cholesterol efflux experiments as         in panel C. n=6; *, p<0.05; **, p<0.01.

FIG. 4. Role of ABCA1/G1 in Angiogenesis and in Mediating Effects of AIBP

-   -   (A-F) Angiogenic defects in ABCA1/G1 morphants. One-cell stage         embryos were injected with 8 ng of control MO, 8 ng AIBP2 MO, or         4 ng ABCA1 MO+4 ng ABCG1 MO. Three days later, the morphants         were analyzed by confocal microscopy. Images of SeA (A) and         SIV (D) are shown. Scale, 50 μm. Graphs show number and length         of SeA ectopic branches (B and C) and SIV ectopic sprouts (E         and F) from 5-12 embryos.***, p<0.001.     -   (G-H) Rescue of the effect of AIBP2 overexpression in ABCA1/G1         morphants. (G) One cell stage embryos were injected with 2 nl of         100 ng/ml myog-AIBP2-mCherry plasmid, myog-AIBP2-mcherry plasmid         together with ABCA1/G1 MO, or myog-mCherry. Two days later, the         embryos were analyzed by confocal microscope. The arrow points         to impaired migration of an SeA at the site of AIBP2-mCherry         expression. Scale, m. Abnormal SeA formation was quantified in         8-16 embryos per group (H). **, p<0.01.     -   (I) Diagram representing the hypothesis that somite expression         of AIBP2 accelerates lateral cholesterol efflux from growing EC         and thereby prevents its ectopic sprouting/branching.

Materials and Methods

Zebrafish: Wild type AB and transgenic Tg(fli1:egfp)y1 and (fli1:negfp)y7 zebrafish lines (13, 35) were kindly provided by Dr. David Traver. Zebrafish were maintained as previously described (36) and all experimental procedures were approved by the UCSD IACUC.

Confocal microscopy: Confocal imaging was carried out as previously described (37). Briefly, anaesthetized zebrafish embryos (treated at 24 hpf with 0.003% PTU) were housed in a sealed chamber (Invitrogen) in a small drop of 0.02% tricaine (Sigma) containing E3 medium. A Nikon C1-si™ confocal microscope was used in a one- or two-cannel mode to detect fluorescence of one or two fluorophores in the same embryo. In the green channel, excitation was 488 nm for EGFP, and in the red channel, 561 nm for mCherry. Z-stacks were acquired with a 1-3 μm step, and images were 3D rendered and analyzed using IMARIS® software (Bitplane). All 3D reconstructions were performed with the same threshold settings.

Quantitative analysis of cell sprouts in segmental arteries (SeA) and subintestinal veins (SIV): To determine changes in segmental artery cell sprouts in embryos injected with MOs targeting AIBP2 and ABCA1/G1, we counted abnormal projections in 4 to 6 pairs of segmental arteries in adjacent somite boundaries in each zebrafish. For each set of injections, 15 embryos (i.e. 60-90 sprouts) were examined Values were expressed as a number of ectopic sprouts per SeA. To examine sprouts in SIV, only the sprouts moving in the ventral direction out of the SIV were counted. Values were expressed as a number of sprouts per zebrafish.

Quantification of cell numbers in segmental artery sprouts: To measure cell proliferation, we counted number of cells (nuclei) in four SeAs in adjacent somite boundaries of Tg(fli1:negfp)y7 zebrafish in which EGFP is expressed only in the nuclei of endothelial cells (35, 39). For each set of injection, 8-9 embryos were examined Values were then calculated as percentages of the total number of sprouts having a certain number of segmental artery cells.

Morpholino oligonucleotide injections: To knock down gene expression, 8 ng of morpholino oligonucleotides (MO; synthesized by GeneTools) were injected into one-cell stage embryos. A control MO was derived from the AIBP2 sequence, with 5 mismatched oligonucleotides.

Control MO: (SEQ ID NO: 1) TGAGCTTCATGTTCATTTATTCCGC; AIBP2 MO: (SEQ ID NO: 2) TGTGGTTCATCTTGATTTATTCGGC; AIBP2 splicing MO: (SEQ ID NO: 3) TGTTGAGTGTCAGACAAACCTTGGT; AIBP1 MO: (SEQ ID NO: 4) TCTGTATTCAAATCAGACGCTCAGT; ABCA1 MO: (SEQ ID NO: 5) AACCCAACTGAGTGGAGACAGCCAT; ABCG1 MO: (SEQ ID NO: 6) AAAAGGCTGCCATGAGACATGCCAT.

Whole mount in situ hybridization: WISH was carried out as described (38). All WISH probes (39, 40), except AIBP2, were kindly provide by Dr. David Traver (UCSD). Briefly, WT embryos or morphants at indicated developmental stages were fixed with 4% paraformaldehyde and permeabilized with 10 μg/ml protease K (Roche). Subsequently, the embryos were pre-hybridized at 70° C. for 4-6 hours, and then hybridized with a digoxigenin-labeled AIBP2 antisense probe at 65° C. overnight. Both the control sense and probe anti-sense RNA were directly synthesized from AIBP2 full length gene using a Roche T7, T3 or SP6 RNA synthesis kit. After extensive wash, hybridized RNA was detected by immunohistochemistry using an alkaline phosphatase-conjugated antibody against digoxigenin (Roche) and a chromogenic substrate Nitro Blue Tetrazolium (NBT) and 5-Bromo 4-Chloro3Indolyl Phosphate BCIP (38).

Cloning of zebrafish and human AIBP, recombinant protein expression and purification, and antibody production: Zebrafish AIBP2 and AIBP1 were cloned from zebrafish brain cDNA using primers:

for AIBP 1 (SEQ ID NO: 7) CCGGAATTCCATGTTGGGGGTTCGAGCTCTG(5′) and (SEQ ID NO: 8) CGCGGATCCTCAGTTGAGCTGAAACACACACTC(3′); and for AIBP2 (SEQ ID NO: 9) CCGGAATTCCGCCACCATGAACCACAGCTCCAACG(5′) and (SEQ ID NO: 10) CGCGGATCCCGCAGTTCTATAATACATTCTGTGC(3′).

The fragments were cloned in frame into pFLAG-CMV (Sigma). Human AIBP was cloned from 293 cell cDNA using primers:

(SEQ ID NO: 11) CGGAATTCCATGTCCAGGCTGCGGGCGCTGCTGGGCCTCG(5′), (SEQ ID NO: 12) CGGGGTACCTCACTGCAGACGATAGACACACTC(3′).

For expression of AIBP proteins, the genes were cloned in frame into pHUE vector (41) (kindly provided by Tracy Handel), and expressed in BL21 DE3 competent cells (Invitrogen) and purified with an Ni-NTA agarose resin column (Qiagen). Deubiquitinase (DUB) expressed in pHUE was used as a negative control in experiments with recombinant AIBP. To produce an AIBP2 antibody, 500 μl of 200 μg of recombinant AIBP2 was mixed with 500 μl complete Freund's adjuvant (Sigma) and injected subcutaneously into a guinea pig. The guinea pig was boosted one month later with a subcutaneous injection of 500 μl antigen mixed with incomplete Freund's adjuvant (Sigma), followed by 2 more boosts (intramuscular injections of 200 μg antigen in 1 ml PBS, 2 weeks apart). Post-immune plasma was compared with pre-immune plasma from the same animal and used in western blot to detect AIBP2 in a zebrafish lysate. The specificity of the antibody was confirmed by adding excess of recombinant AIBP2 to the antibody, which prevented its binding to a specific band on western blot.

Transplantation experiments: Cell transplantation was performed as described (39), with different combinations of donors and recipients as indicated in FIG. 1. Donor embryos were of the Tg(fli1:egfp)^(y1) origin, and recipient embryos were of the wild type origin. At the one-cell stage, donor Tg(fli1:egfp)^(y1) embryos were injected with rhodamine-labeled dextran (Mini-Ruby, Invitrogen) as a lineage tracer. At the sphere stage (about 4 hpf), embryos were dechorionated by 0.4 mg/ml pronase (Sigma) and transferred to agarose wells (Adaptive Science Tools, Worcester, Mass.). Approximately 20-cells from the margin of a donor embryo were transferred to the margin of a recipient embryo. The recipient embryos were subsequently grown at 28° C. and imaged at 72 hpf. Endothelial cells in chimeric zebrafish originating from donor embryos were visualized by their green fluorescence using a Leica M165FC™ fluorescent stereoscope. For detailed analysis images were captured using a Nikon C1-si™ confocal microscope. Numbers of ectopic branches in each fluorescent SeA were counted.

Microarray: Fifty control MO or AIBP2 MO embryos at 12 and 24 hpf were dechorionated, and total RNA was extracted with a Qiagen's RNEASY KIT™ (RNeasy Kit), with modifications. In brief, dechorionated embryos were immediately placed in RNALATER™ (RNAlater) (Qiagen), and incubated for 10 min at room temperature. The embryos were then homogenized in TRIZOL™ (Invitrogen) for 30 to 60 seconds on ice, chloroform was added and the sample was vortexed for 30 sec, incubated for 5 min at room temperature and centrifuged for 30 min at 13,000 rpm at 4° C. The upper layer was transferred to a new tube, supplemented with 70% ethanol and mixed thoroughly. The sample was then processed according to the RNEASY KIT™ manual. The microarray analysis was performed in UCSD's Biogem Core facility using a Zebrafish (V3) Gene Expression Microarray, 4×44k™, Cat# G2519F-026437 from Agilent.

Real time PCR: Real time PCR was performed using a ROTOR GENE Q™ qPCR machine (Qiagen). Real time PCR master mix PLATINUMR™ (PlatinumR™) SYBRR Green qPCR SUPERMIX™ was from Invitrogen. The primers were synthesized by IDT. The PCR program was: 50° C. for 2 minutes (UDG incubation), 95° C. for 2 minutes, 40 cycles of: 95° C. for 15 seconds, 60° C. for 1 minute.

Primer sequences:

Fli1a (F): (SEQ ID NO: 13) CTTGGCACGTTGCCTTGATAAG, Fli1 (R): (SEQ ID NO: 14) CCTTCATATCTGAGAGTGATCCC; Flk1 (F): (SEQ ID NO: 15) TCCACGAGGGTGGGCAGTCA, Flk1(R): (SEQ ID NO: 16) AGACGGGTGGTGTGGAGTAACGA. Tie2 (F): (SEQ ID NO: 17) GCGATGGATGGCAATAGAGT, Tie2 (R): (SEQ ID NO: 18) CGACAGCAGGATCTGAGAGA; Kdr (F): (SEQ ID NO: 19) TGCCCACATGGAGCTGCTAGCA, Kdr (R): (SEQ ID NO: 20) TGTGGCACATTCAACCACATGAGC.

Immunoblot: Zebrafish were lysed on ice with a lysis buffer (50 mM Tris-HCl, pH 7.5, 4 mM sodium deoxycholate, 1% Triton X 100, 150 mM NaCl, 1 mM EDTA, and protease inhibitor cocktail from Sigma). Protein content was determined with a DC protein assay kit (BioRad) and equal protein amounts of the cell lysates were run on a 4-12% Bis-Tris SDS-PAGE with MOPS buffer (Invitrogen) and then transferred to a PVDF membrane (Invitrogen). The blots were probed with appropriate antibodies against specific phosphorylated and non-phosphorylated proteins (Cell Signaling Technology), secondary antibodies conjugated with HRP and developed using a Super Signal West Dura™ substrate (Pierce).

Filipin staining: Zebrafish were fixed with 4% paraformaldehyde overnight at 4° C. The fixed fish were incubated overnight with 0.05% filipin (Sigma) in PBS with 1% sheep serum, washed 3 times with PBS and mounted in glycerol. Images were captured and quantified with a Leica M165FC™ fluorescent stereoscope.

Total lipid extraction and free cholesterol measurements: Total lipid was extracted from zebrafish embryos as we previously described (42). In brief, trunk/tail segments were dissected from fifty 24 hpf embryos and pooled together. The tissue was

homogenized and supplemented with 50 μg stigmasterol, an internal standard to control for recovery of extracted sterols. Total lipid extraction was performed with 1:2 methanol/dichloromethane. No saponification of cholesteryl esters was performed because the goal of this study was to measure free cholesterol, the form of cholesterol transferred from the cells via ABC transporters to ApoA-VHDL. Cholesterol and stigmasterol were measured with a Shimadzu GC-2014TH gas chromatograph using a 30 m×0.25 mm (i d) ZB-SHT™ inferno capillary column, film thickness 0.2 μm (Phenomenex). Cholesterol levels were normalized to protein and then to the levels in embryos injected with control MO.

Cholesterol efflux: A cholesterol efflux assay was performed as described (28, 43), with modifications. In brief, human umbilical vein endothelial cells (HUVEC; from ATCC) were loaded with 2 μCi/ml 3H-cholesterol in 5% fetal bovine serum-supplemented EBM media (Lonza). Twenty-four hours later, cells were washed and equilibrated for 1 hour with DMEM containing 0.2% bovine serum albumin (BSA). The cells were washed with PBS and cholesterol efflux was initiated by the addition of 0.2% BSA/DMEM with 50 μg/ml HDL3 (isolated from normolipidemic human plasma by ultracentrifugation), in the presence or absence of 0.2 μg/ml AIBP2, AIBP1, or hAIBP. Replacing AIBP with deubiquitinase (DUB) was used as a negative control. Background, non-specific release of 3H-cholesterol was measured in 0.2% BSA/DMEM, in the absence of HDL or any other protein. After 1 to 6 hours of incubation, the medium was collected and counted in a multipurpose liquid scintillation counter LS 6500™ (Beckman Coulter). The cells were extracted with 2-propanol, the lipid extract evaporated under argon gas, resuspended in 2-propanol and then added to SCINTIVERSE BD COCKTAIL™ (Fisher), and counted. Cholesterol efflux was expressed as a percentage of 3H counts in the medium compared to total 3H counts in the cells and the medium. Background, non-specific release of 3H from the cells was subtracted.

ABCG1 knockdown: Both negative control and ABCG1 siRNA oligonucleotides were from Ambion. HUVEC were plated in 24-well plates at 1×10⁵ cells/well and transfected with 20 nM siRNA using SUPERFECT TRANSFECTION REAGENT™ (Qiagen) as described in the manufacturer's protocol. Two days after transfection, cells were washed and used in an efflux assay. Two additional wells of transfected cells were used to confirm ABCG1 knockdown in western blot using an antibody from Novus Biologicals.

In vitro angiogenesis assay: The angiogenesis assay was carried out as described in (44). Growth factor reduced MATRIGEL™ (BD Biosciences) was thawed at 4<C overnight and diluted with an equal volume of serum-free EBM medium (Lonza). Each well of pre-chilled 96-well plates was coated with 50 μl diluted Matrigel and incubated at 37<C for 1 hour. HUVECs were serum-starved for 2 days and then pre-incubated for 4 hours with 50 μg/ml HLD3, 0.1 μg/ml hAIBP, or 50 μg/ml HLD3+0.1 μg/ml hAIBP. Cells were harvested and added to Matrigel-coated plates at 1˜104 cells per well in EBM supplemented with 2% LPDS, in the presence or absence of 20 ng/ml VEGF. Tubular structures were imaged following a 12 hour incubation with a phase contrast microscope.

Example 2 Exemplary Compounds and Methods of the Invention Regulate Cholesterol Transport Cholesterol Efflux

This example describes and demonstrates exemplary activities, and the efficacy, of compounds and methods of the invention. We investigated whether human AIBP (hAIBP) would have any effect on cholesterol efflux from EC to HDL.

In human umbilical vein endothelial cells (HUVEC), ABCG1 is a major cellular transporter responsible for cholesterol efflux to HDL (10, 11). In the presence of hAIBP, cholesterol efflux from HUVEC to HDL3 was increased 2-fold, and the ABCG1 deficiency completely abrogated this effect (FIG. 1a ). Increased cholesterol efflux in the presence of hAIBP was observed within first 4 hours of incubation and then, at 6 hours, leveled off with the cholesterol efflux to HDL3 in the absence of hAIBP (Supplementary FIG. 2).

To investigate the role of AIBP/HDL-mediated cholesterol efflux in angiogenesis, we added hAIBP, in the presence or absence of HDL3, to HUVEC and stimulated cells with VEGF. hAIBP and HDL3 added separately did not affect EC tube formation, but together they completely blocked angiogenesis (FIG. 1b ). If the hAIBP/HDL inhibition of angiogenesis is the consequence of accelerated cholesterol efflux, then this effect should depend on the presence of the cholesterol transporter ABCG1. Indeed, knockdown of ABCG1 in HUVEC rescued VEGF-induced angiogenesis from the hAIBP inhibition (FIG. 1c ). ABCG1-mediated depletion of cholesterol from plasma membrane disrupts cholesterol- and sphingolipid-rich membrane microdomains 12, often designated as lipid rafts, and affects surface receptor signalling (13,14). Adding hAIBP/HDL3 significantly reduced lipid raft content in HUVEC (FIG. 1d ). Accordingly, VEGF-induced phosphorylation of VEGFR2, Akt, FAK, Src and to a lesser degree of ERK1/2 were reduced in HUVEC pretreated with hAIBP/HDL3, but not with hAIBP or HDL3 alone (FIG. 1e ). Consistent with the effect on VEGF signalling, HUVEC migration toward the VEGF cue was significantly reduced in hAIBP/HDL3-treated cells (Supplementary FIG. 3). These results demonstrate that hAIBP/ABCG1-mediated cholesterol efflux to HDL disrupts lipid rafts and VEGF signalling in HUVEC and thereby inhibits VEGF-induced angiogenesis.

AIBP is evolutionary conserved from Drosophila to zebrafish to mouse and human (Supplementary FIG. 4a ). Zebrafish have two AIBP genes, apoa1bp1 and apoa1bp2 (Supplementary FIG. 4b ). Both proteins bound to human ApoA-I and to the HDL in human plasma (Supplementary FIG. 5a-c ). In a HUVEC cholesterol efflux assay, zAibp2 was as effective in promoting cholesterol efflux to HDL3 as hAIBP, with zAibp1 having a lesser effect (Supplementary FIG. 6).

To demonstrate that the AIBP effect on angiogenesis is not restricted to HUVEC, we tested both hAIBP and zAibp2 in an ex vivo aortic ring angiogenesis assay. A cluster of HEK293 cells producing either hAIBP or zAibp2 was placed in close proximity to a mouse aortic ring, and then angiogenesis was stimulated with addition of VEGF. Both hAIBP and zAibp2 completely abrogated aortic ring neovascularization (FIG. 1f ).

To investigate whether zAibp2 plays any role in zebrafish cholesterol metabolism, we injected one-cell stage embryos of wild type zebrafish with antisense morpholino oligonucleotides (MO) targeting zaibp2 translation sites or with scrambled control MO. At 24 hours post fertilization (hpf) the embryos were stained with filipin, a fluorescent dye that specifically binds to free (unesterified) cholesterol. The filipin fluorescence was increased in the body of zaibp2 morphants (excluding the yolk) compared to controls (FIG. 2a, b ). These results were confirmed with direct gas chromatography measurements of free cholesterol in lipid extracts from pooled de-yolked embryos (FIG. 2c ). Increased levels of free cholesterol measured by two independent techniques suggest that zAibp2 regulates cholesterol metabolism and, thus, based on the in vitro results shown in FIG. 1, may control angiogenesis. Indeed, injection of MO targeting translation sites of zaibp2 into one-cell stage embryos of Tg(fli1-egfp)y1zebrafish, which express EGFP in EC15, resulted in remarkable dysregulation of angiogenesis.

Profound ectopic branching of both segmental arteries (SeA) and subintestinal veins (SW) were found in zaibp2 morphants (FIG. 2d, e ), whereas no obvious abnormalities were observed in zaibp1 morphants (data not shown). Similar results were observed with the MO targeting a zaibp2 splicing site (Supplementary FIG. 7). The zaibp2 knockdown was validated in western blot (see FIG. 3D). The ectopic branching of SeAs in zaibp2 morphants was partially rescued by forced expression of zaibp2 mRNA lacking the MO target site (FIG. 2f ).

Next, the tissue distribution of zaibp2 in embryos at different developmental stages was examined by whole embryo in situ hybridization (WISH). zaibp2 expression was detected as early as 20 min post fertilization and, notably, zaibp2 mRNA at 24 hpf was expressed within the somites (FIG. 3a ). The zaibp2 expression pattern resembles that of type 3 semaphorins, non-cell autonomous repellent cues that guide the patterning of developing SeAs via endothelial-specific PlexinD1 receptors (16,17). To determine the cell autonomy of zAibp2 effect on angiogenesis, we performed cell transplantation experiments, using Tg(fli1:egfp)^(y1) donors and non-transgenic recipients. Fluorescent EC from wild type donors found in non-fluorescent zaibp2 morphants displayed excessive branching and filopodial projections (FIG. 3b ).

In contrast, fluorescent EC from zaibp2 morphant donors found in wild type recipients had normal morphology. In a gain-of-function experiment, ectopic expression of zAibp2 (and DsRed as a fluorescent tracer) in somites, driven by myog:Ga14 and bi-directional 10×UAS promoters, inhibited SeA sprouting from the dorsal aorta and normal growth of sprouted SeA (FIG. 3c ). These results demonstrate a role for zAibp2 as a repellent molecule whose function depends on the milieu surrounding EC but not on zAibp2 expression in the EC themselves.

The impact of the loss of zAibp2 on molecular pathways involved in angiogenesis was examined in control and zaibp2 morphants using microarrays. Genes indicative of enhanced cell proliferation and genes involved in angiogenesis, such as tie2, vegfr2, vegfr3 and fli1, were upregulated in zaibp2 morphants (Supplementary FIG. 8). Increased expression of genes involved in angiogenesis was confirmed by both qRT-PCR and WISH (FIG. 3d and Supplementary FIG. 9). Lysates of zaibp2 knockdown embryos displayed robust phosphorylation of Akt, VEGFR2 and Src, but not of ERK1/2 (FIG. 3e and Supplementary FIG. 10). These results demonstrate that the molecular pathways that orchestrate angiogenesis are dramatically affected by zaibp2 knockdown.

To further test the hypothesis that effective cholesterol efflux is required for normal angiogenesis, we knocked down zebrafish abca1 and abcg1. Expression of abca1 and abcg1 has been found in zebrafish and, as in mammals, it is induced by liver X receptor agonists (18,19). As in zaibp2 morphants, lysates of zebrafish injected with abca1+abcg1 MO displayed increased levels of phosphorylated Akt, VEGFR2 and Src (FIG. 3e ). The abcg1 knockdown was confirmed as shown in Supplementary FIG. 11.

Further, we found that the loss of Abca1 and Abcg1 in zebrafish closely reproduced the vascular developmental defects of zaibp2 morphants, in both SeA and, markedly, in SIV vascular beds (FIG. 4a ). As expected, abca1/abcg1 morphants had higher levels of free cholesterol than control larvae (Supplementary FIG. 12).

In agreement with the zaibp2 knockdown results, overexpression of zAibp2-mCherry in somites resulted in inhibition of SeA growth and incomplete SeA angiogenesis (FIG. 4b ). Remarkably, the inhibition of SeA formation induced by ectopic zAibp2-mCherry expression was rescued by knocking down abca1/abcg1. These results provide additional evidence that expression of zAibp2 limits blood vessel growth and also suggest that Abcg1 and/or Abca1 are required for zAibp2 function in cholesterol efflux. The latter conclusion agrees with the results that ABCG1 is required for hAIBP inhibition of HUVEC tube formation (FIG. 1a ). The findings of this work demonstrate that effective cholesterol efflux is necessary for proper sprouting, branching and growth of blood vessels in vivo.

Furthermore, we identified Aibp as a novel factor controlling zebrafish angiogenesis and suggested the mechanism in which Aibp functions as a non-cell autonomous positive regulator of cholesterol efflux from EC. Because in 24 hpf zebrafish Aibp is highly expressed in somites (FIG. 3a ), it is likely that it promotes lateral cholesterol efflux from EC of growing SeA, reduces lipid rafts and inhibits local VEGF signaling, thus preventing ectopic SeA growth through the somites (Supplementary FIG. 1). Because VEGFR signaling requires receptor dimerization (20), which occurs in lipid raft microdomains (13), AIBP-mediated cholesterol efflux and associated reduction of lipid rafts provide a new level of paracrine regulation for the VEGFR pathway.

In addition, a role for AIBP in facilitating Notch and/or PlxnD 1 signaling, key negative regulators of angiogenesis (21-23), cannot be excluded. The role of cholesterol efflux mechanisms in protecting against endothelial dysfunction, in particular in hypercholesterolemic animals prone to development of atherosclerosis, has been reported (11, 24). However, our study is the first to demonstrate the importance of cholesterol efflux in regulation of angiogenesis.

The reports showing modulation of angiogenesis by statins, inhibitors of HMG-CoA reductase, demonstrate that the statins' effects are due to changes in prenylation of signaling proteins, not in cholesterol levels (25, 26). Furthermore, our study for the first time demonstrates the role of AIBP in cholesterol efflux from EC and in vivo angiogenesis. In vitro and ex vivo experiments with hAIBP, HUVEC and mouse aortic tissue imply that the importance of AIBP extends beyond regulation of zebrafish angiogenesis and that AIBP may play a role in mammalian angiogenesis as well.

Interestingly, treatment of rat aortic rings with VEGF significantly reduces Aibp mRNA expression (27), implying that downregulation of AIBP is required for neovascularization. It is noteworthy that the human APOA1BP gene is located at the chromosome 1 locus for familial combined hyperlipidemia and that APOA1BP is differentially expressed in many angiogenic tumors (28, 29).

This invention demonstrates that AIBP functions in embryonic angiogenesis and the results of our ex vivo and in vitro experiments demonstrate the role of AIBP in a variety of pathologic and regenerative processes in humans, from cancer to cardiovascular disease.

Methods Example 2

Human and zebrafish AIBP were cloned, expressed and purified using standard techniques, and used in in vitro cholesterol efflux and angiogenesis assays as described (11, 24, 30). In vivo angiogenesis was monitored in Tg(fli1:egfp)y1 zebrafish in which zaibp2 was ectopically expressed or zaibp2 and/or abca1 and abcg1 were knocked down by antisense MO. VEGFR expression and signaling were detected by qPCR and immunoblot.

See also methods as set forth in Example 1.

REFERENCES Example 2

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LXR Signaling Couples Sterol Metabolism     to Proliferation in the Acquired Immune Response. Cell 134, 97-111     (2008). -   7. Ritter, M., et al. Cloning and characterization of a novel     apolipoprotein A-I binding protein, AI-BP, secreted by cells of the     kidney proximal tubules in response to HDL or ApoA-I. Genomics 79,     693-702 (2002). -   8. Bodnar, J. S., et al. Positional cloning of the combined     hyperlipidemia gene Hyplipl. Nat Genet 30, 110-116 (2002). -   9. Ma, K. N., et al. Biochemical and structural characterization of     apolipoprotein A-I binding protein, a novel phosphoprotein with a     potential role in sperm capacitation. Endocrinology 149, 2108-2120     (2008). -   10. O'Connell, B. J. Cellular Physiology of Cholesterol Efflux in     Vascular Endothelial Cells. Circulation 110, 2881-2888 (2004). -   11. Terasaka, N., et al. ABCG1 and HDL protect against endothelial     dysfunction in mice fed a high-cholesterol diet. Journal of Clinical     Investigation 118, 3701-3713 (2008). -   12. Mendez, A. J., Lin, G., Wade, D. P., Lawn, R. M. & Oram, J. F.     Membrane lipid domains distinct from cholesterol/sphingomyelin-rich     rafts are involved in the ABCA1-mediated lipid secretory pathway. J     Biol Chem 276, 3158-3166 (2001). -   13. Simons, K. & Ikonen, E. Functional rafts in cell membranes.     Nature 387, 569-572 (1997). -   14. Paila, Y. D. & Chattopadhyay, A. Membrane cholesterol in the     function and organization of Gprotein coupled receptors. Subcell     Biochem 51, 439-466 (2010). -   15. Lawson, N. D. & Weinstein, B. M. In vivo imaging of embryonic     vascular development using transgenic zebrafish. Dev Biol 248,     307-318 (2002). -   16. Gu, C. Semaphorin 3E and Plexin-D1 Control Vascular Pattern     Independently of Neuropilins. Science 307, 265-268 (2005). -   17. Torres-Vázquez, J., et al. Semaphorin-Plexin Signaling Guides     Patterning of the Developing Vasculature. Developmental Cell 7,     117-123 (2004). -   18. Futter, M., et al. Wild-type but not mutant huntingtin modulates     the transcriptional activity of liver X receptors. J Med Genet 46,     438-446 (2009). -   19. Thisse, B., Pfumio, S., Fiirthauer, M., Loppin B., Heyer, V.,     Degrave, A., Woehl, R., Lux, A., Steffan, T., Charbonnier, X. Q. and     Thisse, C. Expression of the zebrafish genome during embryogenesis     (NIH RO1 RR15402). ZFIN Direct Data Submission (http://zfin.org)     (2001). -   20. Ruch, C., Skiniotis, G., Steinmetz, M. O., Walz, T. &     Ballmer-Hofer, K. Structure of a VEGFVEGF receptor complex     determined by electron microscopy. Nat Struct Mol Biol 14, 249-250     (2007). -   21. Lawson, N. D. & Weinstein, B. M. Arteries and veins: making a     difference with zebrafish. Nat Rev Genet 3, 674-682 (2002). -   22. Adams, R. H. & Alitalo, K. Molecular regulation of angiogenesis     and lymphangiogenesis. Nat Rev Mol Cell Biol 8, 464-478 (2007). -   23. Herbert, S. P. & Stainier, D. Y. Molecular control of     endothelial cell behaviour during blood vessel morphogenesis. Nat     Rev Mol Cell Biol 12, 551-564 (2011). -   24. Whetzel, A. M., et al. ABCG1 Deficiency in Mice Promotes     Endothelial Activation and Monocyte-Endothelial Interactions.     Arteriosclerosis, Thrombosis, and Vascular Biology 30, 809-817     (2010). -   25. Kureishi, Y., et al. The HMG-CoA reductase inhibitor simvastatin     activates the protein kinase Akt and promotes angiogenesis in     normocholesterolemic animals. Nature medicine 6, 1004-1010 (2000). -   26. Choi, J., et al. Aplexone targets the HMG-CoA reductase pathway     and differentially regulates arteriovenous angiogenesis. Development     138, 1173-1181 (2011). -   27. Aplin, A. C., Gelati, M., Fogel, E., Carnevale, E. &     Nicosia, R. F. Angiopoietin-1 and vascular endothelial growth factor     induce expression of inflammatory cytokines before angiogenesis.     Physiol Genomics 27, 20-28 (2006). -   28. Skawran, B., et al. Gene expression profiling in hepatocellular     carcinoma: upregulation of genes in amplified chromosome regions.     Mod Pathol 21, 505-516 (2008). -   29. Lenburg, M. E., et al. Previously unidentified changes in renal     cell carcinoma gene expression identified by parametric analysis of     microarray data. BMC Cancer 3, 31 (2003). -   30. Carmona, G., et al. Role of the small GTPase Rap1 for integrin     activity regulation in endothelial cells and angiogenesis. Blood     113, 488-497 (2009).

Figure Legends Example 2 FIG. 1. Role of AIBP in Cholesterol Efflux from EC and In Vitro Angiogenesis

-   -   a, Effect of ABCG1 knockdown on hAIBP mediated-cholesterol         efflux. HUVEC were transfected with control or ABCG1 siRNA. The         ABCG1 knockdown was confirmed in western blot. HUVEC were         preloaded with 3H-cholesterol and then incubated for 1 hour with         50 mg/ml HDL3 in the presence or absence of 0.2 mg/ml hAIBP. The         media and cell lysates were collected and 3H counts were         measured. Efflux was measured as the 3H counts in the medium         divided by the sum of 3H counts in the medium and the cell         lysates. Mean±SE; n=6; *, p<0.05; **, p<0.01.     -   b, Effect of hAIBP and HDL3 on EC tube formation. HUVEC were         preincubated with 50 μg/ml HDL3, 0.1 μg/ml hAIBP, or 50 μg/ml/ml         HDL3+0.1 μg/ml hAIBP for 4 hours. Cells were then seeded on         Matrigel, in the presence or absence of 20 ng/ml VEGF, and         imaged following a 12 hour incubation. Scale, 100 μm. The length         of EC tubes was measured. Mean±SE; n=5. *, p<0.05; ***, p<0.001.     -   c, Requirement for ABCG1 in hAIBP inhibition of angiogenesis.         HUVEC were transfected with control or ABCG1 siRNA and used in a         EC tube formation assay as in panel C. Mean±SE; n=6. ***,         p<0.001.     -   d, Effect of hAIBP and HDL3 on lipid rafts. HUVEC were         preincubated with 50 μg/ml HDL3, 0.1 μg/ml hAIBP, or 50 μg/ml/ml         HDL3+0.1 μg/ml hAIBP for 4 hours. Cells were stained for nuclei         (blue, DAPI) and for lipid rafts (red, cholera toxin B (CTB) and         anti-CTB antibody), and the area of lipid rafts per cell were         measured. Mean±SE; n=10. **, p<0.01; #, p=0.08.     -   e, Effect of hAIBP on VEGF signaling. HUVEC were preincubated         with 50 μg/ml HDL3, 0.2 μg/ml hAIBP, or 50 μg/ml HDL3+0.2 μg/ml         hAIBP for 4 hours, followed by a 20 min stimulation with 50         ng/ml VEGF. Cells were lysed, and the lysates run on SDS-PAGE         and probed as indicated.     -   f, Aortic ring angiogenesis assay. Aorta was isolated from a         C57BL6 mouse and sliced into 1 mm wide rings. The aortic rings         were embedded in Matrigel. HEK293 cells expressing zAibp2 or         hAIBP were inserted approximately 0.5 mm away from the aortic         ring, and the plates were incubated with 10 ng/ml VEGF for 7         days. Images show the edge of the aortic rings facing the HEK293         cell plaques. Immunoblots show expression of hAIBP and zAibp2 in         HEK293 cells.

FIG. 2. Effect of AIBP Deficiency on Zebrafish Cholesterol Metabolism and Angiogenesis

-   -   a-c. Free cholesterol levels in zaibp2 morphants. a, One-cell         stage AB zebrafish embryos were injected with 8 ng of either         control MO or zaibp2 MO targeting ATG translation site. Twenty         four hpf control and zaibp2 morphants were fixed and stained         with filipin to detect free cholesterol in embryos. Note the         yolks are artificially masked on the images.     -   b. Intensities of filipin fluorescence in images shown in panel         A (without yolks) were quantified in 12-15 embryos per group. *,         p<0.05.     -   c. At 24 hpf, the trunk area (without yolk and head) was         dissected and total lipids were extracted. Free cholesterol         levels were determined by GC. The cholesterol levels were         normalized to the protein content and then to the values in         control MO embryos. 50-70 embryos were pooled for each sample.         The data are from 3-4 independent experiments. *, p<0.05.     -   d-f. Angiogenic defects in zaibp2 morphants. One-cell stage         Tg(fli1:egfp)y1 zebrafish embryos were injected with 8 ng of         either control MO or zaibp2 MO targeting ATG translation site.     -   d. Low magnification, bright field images showing the area of         fluorescent imaging of SeA (white box) and     -   SIV (red box).     -   e. The images of SeA in 30 hpf embryos (top row), and SeA         (images 1-2) and of SIV (3-4) in 3 dpf embryos. Arrows point to         dysregulated sprouts. Scale in all images, 25 m.     -   f. Quantification of the number of embryos with normal and         abnormal angiogenesis (SeA with ectopic branching) resulting         from injection of control or zaibp2 MO. The abnormal         angiogenesis was partially rescued by co-injection of 40 μg of         zaibp2 mRNA lacking the MO targeting site. 100-149 embryos per         group. ***, p<0.001.

FIG. 3. Effect of AIBP Deficiency and Ectopic Overexpression on Zebrafish Angiogenesis and VEGF Signaling

-   -   a, Tissue distribution of zaibp2 mRNA in zebrafish embryo.         Embryos at different developmental stages were fixed and WISH         was performed with an antisense zaibp2 probe. Scale, 100 nm.     -   b, Mosaic expression analysis of EC branching in WT and zaibp2         knockdown embryos. At 4 hpf, cells were isolated from donor         embryos and transplanted into recipient embryos (details of         donor and recipient embryos are depicted above images). Three         days after transplantation, the recipient embryos were analyzed.         Red arrowheads indicate aberrant ectopic branches/sprouts.         Scale, 20 nm. The graph quantifies numbers of ectopic         branches/filopodial projections per SeA. 8-16 embryos per group.         ***, p<0.001.     -   c, Ectopic expression of zAibp2 inhibits SeA growth. One-cell         stage Tg(fli1:egfp)y1 embryos were injected with 2 nl of 100         ng/μl DNA constructs for myog:Ga14, DsRed:10×UAS:zAibp2 or         myog:Ga14, DsRed:10×UAS expression. Images were captured at 30         hpf. Arrowheads point to aberrant SeA growth, close to the sites         of DsRed (and zAibp2) expression.     -   d, Expression of genes involved in angiogenesis. Total RNA was         isolated from 24 hpf control and zaibp2 morphants and qRT-PCR         was performed. The results are normalized to actin. The results         are from a pool of 50 embryos per group, 3-4 replicates.***,         p<0.001; **, p<0.01; *, p<0.05.     -   e, Phosphorylation of signaling proteins. Lysates of 24 hpf         control (8 ng control MO), zaibp2 (8 ng zaibp2 MO) and abca1/g1         (4 ng abca1 MO+4 ng abcg1 MO) morphants were separated on         SDSPAGE and immunoblotted as indicated.

FIG. 4. Role of Abca1 and Abcg1 in Angiogenesis and in Mediating Effects of Aibp

-   -   a, Angiogenic defects in abca1/g1 morphants. One-cell stage         embryos were injected with 8 ng of control MO, 8 ng zaibp2 MO,         or 4 ng abca1 MO+4 ng abcg1 MO. Images of SeA (30 hpf) and SIV         (3 dpf) are shown. Scale, 50 μm. Graphs show numbers and of SeA         ectopic branches and numbers and length of SW ectopic sprouts         from 5-12 embryos.***, p<0.001.     -   b, Rescue of the effect of zAibp2 overexpression in abca1/g1         morphants. One-cell stage embryos were injected with 2 nl of 100         ng/μl myog:zAibp2-mCherry plasmid, myog:zAibp2-mCherry plasmid         together with abca1/g1 MO, or myog:mCherry plasmid. The arrow         points to an aberrant SeA at the site of zAibp2-mCherry         expression. Scale, 20 m. Abnormal SeA formation was quantified         in 8-16 embryos per group. **, p<0.01.

Example 3 Exemplary Compounds and Methods of the Invention Regulate Cholesterol Transport and Angiogenesis: Control of Angiogenesis by AIBP-Mediated Cholesterol Efflux

This example describes and demonstrates exemplary activities, and the efficacy, of compounds and methods of the invention. This example demonstrates that ApoA-I binding protein (AIBP) accelerates cholesterol efflux from endothelial cells (EC) to HDL and thereby regulates angiogenesis. AIBP/HDL-mediated cholesterol depletion reduces lipid rafts, interferes with VEGFR2 dimerization and signaling, and inhibits VEGF-induced angiogenesis in vitro and mouse aortic neovascularization ex vivo. Aibp regulates the membrane lipid order in embryonic zebrafish vasculature and functions as a non-cell autonomous regulator of zebrafish angiogenesis. Aibp knockdown results in dysregulated sprouting/branching angiogenesis, while forced Aibp expression inhibits angiogenesis. Dysregulated angiogenesis is phenocopied in Abca1/Abcg1-deficient embryos, and cholesterol levels are increased in Aibp-deficient and Abca1/Abcg1-deficient embryos. Our findings demonstrate that secreted AIBP positively regulates cholesterol efflux from EC and that effective cholesterol efflux is critical for proper angiogenesis.

First, we investigated whether human AIBP (hAIBP) had any effect on cholesterol removal from human umbilical vein endothelial cells (HUVEC) in which ABCG1 is a major transporter responsible for cholesterol efflux to HDL^(9,16). In the presence of hAIBP, cholesterol efflux from HUVEC to HDL₃ was increased 2-fold, and the ABCG1 deficiency completely abrogated this effect (FIG. 1a , Supplementary FIG. 2). hAIBP did not promote cholesterol efflux in absence of HDL₃ (Supplementary FIG. 2b ), but the hAIBP binding to HUVEC (Supplementary FIG. 3) increased the overall HUVEC capacity to bind HDL₃ (B_(max)=1.5 vs. 0.8) and the constant of HDL₃ dissociation from HUVEC (K_(d)=1.0×10⁻⁶ M vs. 0.33×10⁻⁶M; FIGS. 1b, c ), thereby creating conditions that would facilitate HDL₃-mediated cholesterol efflux.

To investigate the role of AIBP/HDL-mediated cholesterol efflux in angiogenesis, we incubated HUVEC with hAIBP and/or HDL₃ and then stimulated cells with VEGF. hAIBP and HDL₃ added separately did not affect EC tube formation, but together they significantly reduced angiogenesis (FIG. 1d, e and Supplementary FIG. 4). Cholesterol depletion by methyl-β-cyclodextrin (MβCD)¹¹ also inhibited angiogenesis, whereas cholesterol-loaded MβCD, which delivers cholesterol to the cell, promoted angiogenesis (Supplementary FIG. 5). If the hAIBP/HDL₃ inhibition of angiogenesis is the consequence of accelerated cholesterol efflux, then this effect should depend on the presence of the cholesterol transporter ABCG1. Indeed, knockdown of ABCG1 in HUVEC rescued VEGF-induced angiogenesis from the hAIBP/HDL₃ inhibition (FIG. 1f ). Further, we tested both hAIBP and zAibp2 (a zebrafish protein, which will be discussed later) in an ex vivo aortic ring angiogenesis assay. A cluster of HEK293 cells producing either hAIBP, zAibp2 or mCherry (negative control) was placed 0.5 mm from the edge of a mouse aortic ring, and VEGF was added to stimulate angiogenesis. Both hAIBP and zAibp2, but not mCherry, significantly reduced neovascularization of aortic rings isolated from a wild type mouse (FIGS. 1g, h ). Aortic rings from an Abcg1^(−/−) mouse responded to VEGF with a more vigorous angiogenesis, which was not significantly reduced by hAIBP or zAibp2. These results support the hypothesis that cholesterol efflux is necessary for the AIBP-mediated inhibition of angiogenesis.

HDL-mediated depletion of cholesterol from plasma membrane disrupts cholesterol- and sphingomyelin-rich membrane microdomains^(12,13), often designated as lipid rafts, and affects membrane receptor signaling¹¹. We found that hAIBP/HDL₃ reduced the lipid raft content in HUVEC and disrupted cell-surface colocalization of caveolin-1 and VEGFR2 (FIGS. 2a-c and Supplementary FIG. 6). The hAIBP/HDL₃ treatment, similarly to the treatment with MβCD, decreased VEGFR2 and caveolin-1 localization to the lipid raft fraction isolated from cell lysates (FIG. 2d and Supplementary FIG. 7). Many studies suggest that VEGFR2 localization to lipid rafts facilitates VEGFR2 dimerization and endocytosis¹⁴⁻¹⁷, the steps required for VEGF-mediated signaling¹⁸. In our experiments, the hAIBP/HDL₃ treatment reduced VEGF-induced VEGFR2 dimerization and endocytosis as well as phosphorylation of VEGFR2, Akt, FAK, Src and to a lesser degree of ERK1/2 (FIGS. 2e-g and Supplementary FIGS. 8-10). Importantly, subsequent addition of cholesterol partially reversed inhibition of VEGFR2, FAK and Akt phosphorylation in hAIBP/HDL₃-treated cells (Supplementary FIG. 11). Consistent with the effect on VEGF signaling, HUVEC migration toward a VEGF cue was significantly reduced in hAIBP/HDL₃-treated cells (Supplementary FIG. 12). These results demonstrate that hAIBP facilitates cholesterol efflux from HUVEC to HDL and that cholesterol depletion of the plasma membrane disrupts lipid rafts and VEGF signaling and inhibits VEGF-induced angiogenesis.

AIBP is evolutionary conserved from Drosophila to zebrafish to mouse and human (see FIG. 5). Zebrafish have two genes, apoa1bp1 and apoa1bp2, encoding zAibp1 and zAibp2 proteins, respectively (see FIG. 6). The zaibp2 expression in 24-36 hours post-fertilization (hpf) zebrafish embryos shows a clear segmental pattern, colocalizing with the somite marker myod (FIG. 3a and Supplementary FIG. 14). By 48 hpf, when segmental angiogenesis is completed, zaibp2 is no longer expressed in somites.

Both zAibp2 and zAibp1 bound to human ApoA-I and to the HDL in human plasma, but only zAibp2 was effective in promoting cholesterol efflux from HUVEC to HDL₃ (Supplementary FIGS. 15, 16). Zebrafish embryos injected with antisense morpholino oligonucleotides (MO) targeting zaibp2 translation sites had increased levels of free (unesterified) cholesterol, whereas injections of zaibp1 or scrambled control MO did not result in any changes (FIGS. 3b, c and Supplementary FIG. 17). Thus, we focused on zaibp2. Using the polarity-sensitive fluorescent probe Laurdan, we observed a higher membrane lipid order in the areas of growing segmental arteries (SeA) corresponding to tip cells compared to stalk cells (FIG. 3d, e ), suggesting a higher content of lipid rafts in tip cells, which may positively regulate Vegfr2 signaling. The membrane lipid order was increased in the SeA of zaibp2 morphants compared to controls, and the difference between tip and stalk cells was lost. To test the hypothesis that zAibp2-mediated cholesterol efflux regulates membrane order in growing SeA, we injected zaibp2 morphants with human HDL₃ or with BSA. Adding an excess of HDL₃—to promote cholesterol efflux and to override the zAibp2 deficiency—annulled the increase in membrane order in SeA of zaibp2 morphants, and a spatially indiscriminate HDL₃ excess equalized the membrane order in tip and stalk cells. Adding an excess of BSA had no effect on the membrane order in zaibp2 morphants. Lysates of zaibp2 knockdown embryos displayed increased phosphorylation of Vegfr2, Akt and Src, and decreased phosphorylation of Erk1 (FIG. 3f , Supplementary FIGS. 18, 19). These results demonstrate that zAibp2 regulates cholesterol levels, the membrane lipid order, and Vegfr2 signaling controls angiogenesis.

Injection of MOs targeting zaibp2 translation or splicing sites into one-cell stage embryos of Tg(fli1:egfp)^(y1) zebrafish, which express EGFP in EC¹⁹, resulted in remarkable dysregulation of angiogenesis, with profound ectopic branching of both SeA and subintestinal veins (SIV) (FIG. 3g and Supplementary FIGS. 20, 21). The zaibp2 knockdown was validated in western blot (FIG. 3f ). The ectopic branching of SeAs in zaibp2 morphants was partially rescued by forced expression of zaibp2 mRNA lacking the MO target site (FIG. 3h ).

The zaibp2 expression pattern (FIG. 3a ) resembles that of type 3 semaphorins, non-cell autonomous repellent cues that guide the patterning of developing SeAs via endothelial-specific PlexinD1 receptors²⁰. To determine the cell autonomy of the zAibp2 effect on angiogenesis, we performed cell transplantation experiments, using Tg(fli1:egfp)^(y1) donors. Fluorescent EC from wild type donors found in non-fluorescent zaibp2 morphants displayed excessive branching and filopodial projections, while fluorescent EC from zaibp2 morphant donors found in wild type recipients had normal morphology (FIGS. 4a, b ). In a gain-of-function experiment, overexpression of zAibp2 inhibited SeA sprouting from the dorsal aorta and normal growth of sprouted SeA (Supplementary FIG. 22). These results suggest a role for zAibp2 as a repellent molecule whose function depends on the milieu surrounding EC but not on zAibp2 expression in the EC themselves.

The loss of zAibp2 resulted in increased expression of genes involved in angiogenesis, such as tie2, vegfr2, vegfr3 and fill (Supplementary FIGS. 23-25). Thus, in addition to the zAibp2 effect on the membrane lipid order and Vegfr2 signaling (FIGS. 3d-f ), zAibp2 affects expression of angiogenic genes as well.

To further validate that effective cholesterol efflux is required for normal angiogenesis, we knocked down zebrafish cholesterol transporters abca1 and abcg1^(21,22) and observed higher levels of free cholesterol, increased levels of phosphorylated Akt, Vegfr2 and Src and dysregulated SeA and SIV angiogenesis (FIGS. 3f, 4c and Supplementary FIGS. 19, 26-28), closely reproducing the angiogenesis defects of zaibp2 morphants. Individual knockdown of each abca1 and abcg1 suggested a dominant role of abca1 in embryonic angiogenesis (Supplementary FIG. 29). In contrast to the zaibp2 non-cell autonomous regulation of angiogenesis, fluorescent EC from abca1/abcg1 morphant donors found in wild type recipients displayed excessive SeA branching (FIGS. 4a, b ), confirming that cholesterol efflux from EC is required to restrain ectopic angiogenesis. Overexpression of zAibp2-mCherry in somites resulted in inhibition of SeA growth, which was rescued by knocking down abca1/abcg1 (FIGS. 4d, e ). These results provide additional evidence that expression of zAibp2 limits blood vessel growth and also suggest that zebrafish Abca1- and/or Abcg1-mediated cholesterol efflux is required for the zAibp2 effect on angiogenesis.

Based on our results, we propose that there is an additional level of paracrine regulation for the VEGFR2 pathway in which cholesterol efflux and associated reduction of ordered membrane microdomains/lipid rafts interfere with the VEGFR2 membrane localization, dimerization, endocytosis, and signaling. Because in 24 hpf zebrafish zaibp2 mRNA is highly expressed in somites, but not in the inter-somitic spaces where SeA grow, it is likely that zAibp2-mediated cholesterol efflux inhibits Vegfr2 signaling in a site-specific manner to prevent lateral protrusions from stalk and tip cells and restrains ectopic SeA growth into somites (Supplementary FIG. 1).

The role of cholesterol efflux mechanisms in protecting against endothelial dysfunction, in particular in hypercholesterolemic animals prone to development of atherosclerosis, has been reported^(10,23). However, our study is the first to demonstrate the role of AIBP in promoting cholesterol efflux from EC to HDL and the importance of this mechanism in regulation of angiogenesis. In contrast to the ApoA-I-containing HDL, ApoB-containing LDL and VLDL deliver cholesterol and other lipids to the cell and, thus, are positioned to promote angiogenesis.

Interestingly, a recent paper finds the opposite, that ApoB lipoproteins negatively regulate angiogenesis in zebrafish embryos²⁴. The ApoB protein, but not the lipid components within ApoB-containing lipoproteins, may be responsible for transcriptional regulation of Vegfr1, a soluble decoy receptor for Vegf.

Our experiments uncovered a different, lipid-mediated mechanism in which effective cholesterol efflux is a critical process that ensures proper angiogenesis and Aibp secreted by the surrounding tissues serves as an important negative regulator of angiogenesis.

Figure Legends FIG. 1. Role of AIBP in Cholesterol Efflux from EC and In Vitro Angiogenesis

-   -   a, hAIBP mediated-cholesterol efflux and effect of ABCG1         knockdown. HUVEC were transfected with control or ABCG1 siRNA,         preloaded with ³H-cholesterol and incubated for 1 hour with 50         μg/ml HDL₃ in the presence or absence of 0.2 μg/ml hAIBP. Efflux         was measured as the ³H counts in the medium divided by the sum         of ³H counts in the medium and the cell lysate. Mean±SE; n=6.     -   b and c, Effect of hAIBP on HDL₃ binding to HUVEC. HUVEC were         incubated on ice with the indicated concentration of         biotinylated HDL₃ (b-HDL₃), in the presence or absence of hAIBP         (at a 0.1:50 w/w hAIBP:HDL₃ ratio) and 40× excess of unlabeled         HDL. Each data point is Mean±SE from 3 to 7 independent         experiments. The binding parameters for b-HDL₃/HUVEC binding         were calculated as B_(max)=0.8±0.1 and K_(d)=(0.33±0.10)×10⁻⁶ M         in absence of hAIBP (panel b; R²=0.92, Sy.x=0.1), and         B_(max)=1.5±0.4 and K_(a)=(1.03±0.5)×10⁻⁶ M in the presence of         hAIBP (panel c; R²=0.94, Sy.x=0.1). The differences in B_(max)         and K_(d) values were statistically significant (p<0.01 and         p<0.05, respectively).

d, Effect of hAIBP and HDL₃ on EC tube formation. HUVEC were preincubated with or without 50 μg/ml HDL₃+0.1 μg/ml hAIBP for 4 hours. Cells were then seeded on Matrigel, in the presence or absence of 20 ng/ml VEGF, and imaged following a 12-hour incubation. Scale, 100 μm. e, The length of EC tubes in the experiment shown in panel d. Mean±SE; n=5.

f, Requirement for ABCG1 in hAIBP inhibition of angiogenesis. HUVEC were transfected with control or ABCG1 siRNA and assayed as in panel d. Mean±SE; n=6.

g, Mouse aortic ring angiogenesis assay. Aortic rings from C57BL6 and Abcg^(−/−) mice were embedded in Matrigel. HEK293 cells transiently expressing mCherry, zAibp2 or hAIBP were inserted approximately 0.5 mm away from the aortic ring, and the plates were incubated with 10 ng/ml VEGF for 7 days. Images show the edge of the aortic rings facing the HEK293 cell clusters. Immunoblots show expression of hAIBP and zAibp2 (both detected with a Flag tag antibody) and mCherry in HEK293 cells. h, The length of aortic ring sprouts. Mean±SE; n=10.

In all panels: #, not significant; *, p<0.05; **, p<0.01; ***, p<0.001.

FIG. 2. Effect of AIBP on HUVEC Lipid Rafts, VEGFR2 Localization, Dimerization and Signaling

a, Effect of hAIBP and HDL₃ on lipid rafts. HUVEC were preincubated with 50 μg/ml HDL₃, 0.1 μg/ml hAIBP, or 50 g/ml HDL₃+0.1 μg/ml hAIBP for 4 hours. Cells were stained for nuclei (blue, DAPI) and for lipid rafts (red, cholera toxin B (CTB) and anti-CTB antibody). Scale, 10 μm. b, The area of lipid rafts per cell. Mean±SE; n=10; **, p<0.01; #, p=0.08.

c, Effect of hAIBP and HDL₃ on caveolin-1 and VEGFR2 surface localization. HUVEC were incubated with hAIBP and/or HDL₃ as in 2a, fixed and stained with antibodies to caveolin-1 and VEGFR2. Images were captured using TIRF microscopy and Pearson's coefficient was calculated to assess surface colocalization of VEGFR2 with caveolin-1. Mean±SE; n=38-50; ***, p<0.001.

d, VEGFR2 and caveolin-1 localization to lipid rafts. HUVEC were incubated with 20 μg/ml cholesterol-MβCD for six hours, followed by a 1 hour incubation with or without 50 μg/ml HDL₃+0.1 μg/ml hAIBP, or a 30 min incubation with 10 mM MβCD. HUVEC lysates were separated into lipid rafts and non-lipid rafts fractions by ultracentrifugation, run on SDS-PAGE and blotted with VEGFR2 and caveolin-1 antibodies.

e, Effect of hAIBP and HDL₃ on VEGFR2 dimerization. HUVEC were preincubated with HDL₃ and/or hAIBP as in 1a, followed by a 20 min stimulation with 50 ng/ml VEGF. Cells were treated with a crosslinking reagent, lysed and immunoprecipitated with a VEGFR2 antibody. Monomers and crosslinked dimers of VEGFR2 were visualized on western blot.

f, Effect of hAIBP and HDL₃ on VEGFR2 endocytosis. HUVEC were preincubated with or without 50 μg/ml HDL₃+0.1 μg/ml hAIBP for 4 hours, then stimulated with 50 ng/ml VEGF for 20 min, fixed and stained with antibodies to VEGFR2 (red) and the early endosome marker EEA-1 (green). Yellow and white arrows point to the surface and endosomal localization of VEGFR2. Red dotted line traces cell contour. Scale, 10 μm.

g, Effect of hAIBP and HDL₃ on VEGFR2 signaling. HUVEC were preincubated with HDL₃ and/or hAIBP as in 2a, followed by a 20 min stimulation with 50 ng/ml VEGF. Total cell lysates were run on SDS-PAGE and probed as indicated.

FIG. 3. Effect of Aibp Deficiency on Zebrafish Cholesterol, Membrane Lipid Order, Vegfr2 Signaling and Angiogenesis

a, Tissue distribution of zaibp2 mRNA in zebrafish embryos. Embryos at 24 hpf were fixed and WISH was performed with antisense myod and zaibp2 probes. Scale, 100 μm.

b-c, Free cholesterol levels in zaibp2 morphants. b, Zebrafish embryos were injected with 8 ng of either control MO or zaibp2 MO. Twenty four hpf control and zaibp2 morphants were stained with filipin to detect free cholesterol in embryos. Note the yolks are artificially masked on the images. c, At 24 hpf, the trunk area (without yolk) was dissected, total lipids extracted, and free cholesterol levels determined by gas chromatography (GC). The cholesterol levels were normalized to the protein content and then to the values in control MO embryos. 50-70 embryos were pooled for each sample. Mean±SE; n=4; *, p<0.05. d, Effect of zaibp2 MO on SeA membrane lipid order. Tg(flk1:ras-cherry)^(s896) embryos were injected with control or zaibp2 MO as in 3b and at 24 hpf were stained with 5 μM Laurdan. In the same embryos, confocal images of mCherry fluorescence (bottom images) and the multiphoton images of Laurdan fluorescence (ex 800 nm, em 400-460 nm and 470-530 nm) were captured. The multiphoton results (top row images) are displayed as pseudocolored GP (generalized polarization, a measure of the membrane lipid order) images, cropped to show only the vasculature, i.e. mCherry-positive areas. Scale, 20 μm. e, The graph shows GP values in the areas corresponding to tip and stalk cells of growing SeA and the dorsal aorta (DA) as indicated in 3d. Some one-cell stage embryos were co-injected with 1 nl of 10 mg/ml human HDL₃ or BSA. Note the Y-scale is from 0.2 to 0.5. Mean±SE; n=44-119 SeA in 25-49 embryos; ***, p<0.001.

f, Phosphorylation of signaling proteins. Lysates of 24 hpf control (8 ng control MO), zaibp2 (8 ng zaibp2 MO) and abca1/g1 (4 ng abca1 MO+4 ng abcg1 MO) morphants were separated on SDS-PAGE and immunoblotted as indicated.

g, Angiogenic defects in zaibp2 morphants. One-cell stage Tg(fli1:egfp)^(y1) zebrafish embryos were injected with 8 ng of either control or zaibp2 MO. The images are of SeA in 30 hpf embryos (top row), and SeA (middle row) and of SIV (bottom row) in 3 dpf embryos. Arrows point to dysregulated sprouts. Scale, 25 μm. h, Quantification of the number of embryos with normal and abnormal angiogenesis (SeA with ectopic branching). The abnormal angiogenesis was partially rescued by co-injection of 40 μg of zaibp2 mRNA lacking the MO targeting site. Mean±SE; n=100-149. ***, p<0.001.

FIG. 4. Effect of Aibp and Abca1/Abcg1 Deficiency on Zebrafish Angiogenesis

a, Mosaic expression analysis of EC branching in control, zaibp2 and abca1/abcg1 knockdown embryos. At 4 hpf, cells were isolated from donor embryos and transplanted into recipient embryos. Recipient embryos were analyzed at 3 dpf. Arrows point to aberrant ectopic branches/sprouts. Scale, 25 nm. b, Numbers of ectopic branches/filopodial projections per SeA. Mean±SE; n=8-16. #, not significant; *, p<0.05; ***, p<0.001.

c, Angiogenic defects in abca1/abcg1 morphants. One-cell stage embryos were injected with 8 ng of control MO, 8 ng zaibp2 MO, or 4 ng abca1 MO+4 ng abcg1 MO. Images of SeA (30 hpf) and SIV (3 dpf) are shown. Scale, 50 μm.

d, Knockdown of abca1/g1 cancels the effect of zAibp2 overexpression. One-cell stage embryos were injected with 2 nl of 100 ng/μl myog:zaibp2-mCherry, myog:zaibp2-mCherry+abca1/g1 MO, or myog:mCherry. The arrow points to an aberrant SeA at the site of zAibp2-mCherry expression. Scale, 20 nm. e, Abnormal SeA formation was quantified in 8-16 embryos per group. Mean±SE; *** p<0.001.

Methods

Human and zebrafish AIBP were used in cholesterol efflux, angiogenesis and cellular assays as described^(10,23,25). In vivo angiogenesis was monitored in Tg(fli1:egfp)^(y1) zebrafish with zaibp2 and/or abca1 and abcg1 knockdown or forced zaibp2 expression.

Cloning of Human and Zebrafish AIBP, Recombinant Protein Expression and Purification, and Antibody Production

Zebrafish aibp2 and aibp1 were cloned from zebrafish brain cDNA using primers:

for zaibp1 (SEQ ID NO: 21) CCGGAATTCCATGTTGGGGGTTCGAGCTCTG(5′) and (SEQ ID NO: 22) CGCGGATCCTCAGTTGAGCTGAAACACACACTC(3′); and (SEQ ID NO: 23) CCGGAATTCCGCCACCATGAACCACAGCTCCAACG(5′) and (SEQ ID NO: 24) CGCGGATCCCGCAGTTCTATAATACATTCTGTGC(3′) for zaibp2. The fragments were cloned in frame into pFLAG-CMV4 (Sigma). Human APOA1BP was cloned from HEK293 cell cDNA using primers:

(SEQ ID NO: 25) CCGGAATTCCATGTCCAGGCTGCGGGCGCTGCTGGGCCTCG(5′) and (SEQ ID NO: 26) CGGGGTACCTCACTGCAGACGATAGACACACTC(3′).

For expression of AIBP proteins, the genes were cloned in frame into pHUE vector²⁶ (kindly provided by Tracy Handel), expressed in BL21 DE3 competent cells (Invitrogen) and purified with a Ni-NTA agarose resin column (Qiagen). Deubiquitinase (DUB) expressed in pHUE was used as a negative control in experiments with recombinant AIBP. To produce a zAibp2 antibody, 500 μl (200 μg) of recombinant zAibp2 was mixed with 500 μl complete Freund's adjuvant (Sigma) and injected subcutaneously into a guinea pig. The guinea pig was boosted one month later with a subcutaneous injection of 200 μg antigen mixed with incomplete Freund's adjuvant (Sigma), followed by 2 more boosts (intramuscular injections of 200 μg antigen in 1 ml PBS, 2 weeks apart). Post-immune plasma was compared with pre-immune plasma from the same animal and used in western blot to detect zAibp2 in zebrafish lysates. The specificity of the antibody was confirmed by adding excess of recombinant zAibp2 to the antibody, which prevented its binding to a specific band on the western blot.

Cholesterol Efflux

A cholesterol efflux assay was performed as described^(23,27), with modifications. In brief, human umbilical vein endothelial cells (HUVEC; from ATCC) were loaded with 2 μCi/ml ³H-cholesterol in 5% fetal bovine serum (FBS)-supplemented EBM media (Lonza). Twenty-four hours later, cells were washed and equilibrated for 1 hour with EBM containing 0.2% bovine serum albumin (BSA). The cells were washed with PBS and cholesterol efflux was initiated by the addition of 0.2% BSA/EBM with 50 μg/ml HDL₃ (isolated from normolipidemic human plasma by ultracentrifugation), in the presence or absence of 0.2 μg/ml zAibp2, zAibp1, or hAIBP. Deubiquitinase (DUB), replacing AIBP, was used as a negative control. Background, non-specific release of ³H-cholesterol was measured in 0.2% BSA/EBM, in the absence of HDL or any other protein. After 1 to 6 hours of incubation, the medium was collected and counted in a multipurpose liquid scintillation counter LS 6500 (Beckman Coulter). The cells were extracted with 2-propanol, the lipid extract evaporated under argon gas, resuspended in 2-propanol and then added to ScintiVerse BD Cocktail (Fisher), and counted. Cholesterol efflux was expressed as a percentage of ³H counts in the medium compared to combined ³H counts in the cells and the medium. Background, non-specific release of ³H from the cells was subtracted.

ABCG1 Knockdown

Both negative control and ABCG1 siRNA oligonucleotides were from Ambion. HUVEC were plated in 6-well plates at 5×10⁵ cells/well and transfected with 66.6 nM siRNA using SUPERFECT TRANSFECTION REAGENT™ (Qiagen) as described in the manufacturer's protocol. Two days after transfection, cells were washed and used in an efflux assay. Two additional wells of transfected cells were used to confirm ABCG1 knockdown in western blot using an antibody from Novus Biologicals.

AIBP/HDL₃—HUVEC Binding Assay

hAIBP and HDL₃ were biotinylated with EZ-Link Sulfo-NHS-Biotin according the manufacture's protocol (Thermo Scientific). Binding of biotinylated hAIBP or biotinylated HDL₃ to HUVEC was assessed by a chemiluminescent binding assay as described by Fang et al.²⁸, with modifications. 2×10⁴ HUVEC were seeded into 96-well flat bottom plates in 5% FBS-EBM. After 72 h, plates were washed gently 5 times with PBS using a microtiter plate washer (Dynex Technologies, Chantilly, Va.), blocked with 200 μl of ice-cold 1% BSA-PBS for 30 min on ice, incubated with ice-cold biotinylated proteins (100 μl/well) for 2 h on ice, washed, and fixed with ice-cold 4% paraformaldehyde (PFA) in PBS for 30 min. HUVEC-bound biotinylated hAIBP or HDL₃ were detected with NeutrAvidin-conjugated alkaline phosphatase (Pierce) and LUMIPHOS 530™ (Lumigen, Southfield, Mich.), using a DYNEX™ luminometer (Dynex Technologies). Data were recorded as relative light units counted per 100 ms. All samples were assayed in triplicates. The parameters of hAIBP and HDL₃ bindings to HUVEC (B_(max) and K_(d)) were calculated using a total and non-specific binding algorithm within the GRAPHPAD PRISM 5.0™ software package. The following model was used: H+C⇄ HC, where H is unbound HDL₃, C is cells, and HC is HDL₃ bound to the cells. The equations used for calculating binding parameters were:

[HC] _(specific) =B _(max) ×[H]/([H]+K _(d))

[HC] _(nonspecific) =a+b×[H]

[HC] _(total) =[HC] _(specific) +[HC] _(nonspecific),

where a is background and b is the slope of the linear fit of nonspecific binding. Goodness of fit of non-linear regression was estimated using R² and standard deviation of residuals (Sy.x), expressed in the same units as [H] and B_(max). A molecular mass of 80 kDa was used for the HDL protein (2 molecules of ApoA-I, comprising 70% of the HDL protein mass).

In Vitro Angiogenesis Assay

The angiogenesis assay was carried out as described in reference²⁹. Growth factor reduced Matrigel (BD Biosciences) was thawed at 4° C. overnight and diluted with an equal volume of serum-free EBM medium (Lonza). Each well of pre-chilled 96-well plates was coated with 50 μl diluted Matrigel and incubated at 37° C. for 1 hour. HUVEC were serum-starved and then pre-incubated for 4 hours with 50 mg/ml HLD₃, 0.1 mg/ml hAIBP, or 50 mg/ml HLD₃+0.1 mg/ml hAIBP. Cells were harvested and added to Matrigel-coated plates at 1×10⁴ cells per well in EBM, in the presence or absence of 20 ng/ml VEGF (R&D Systems). Tubular structures were imaged following a 12 hour incubation with a phase contrast microscope.

Free Cholesterol Measurements in HUVEC

HUVEC cholesterol levels were measured in cellular lipid extracts using a colorimetric assay (BioVision) and manufacturer's protocol as described¹⁴. Because in HUVEC non-esterified, free cholesterol comprises more than 95% of total cholesterol¹⁴ and because free cholesterol is the form of cholesterol that is transferred from the cells via ABC transporters to ApoA-VHDL, we only measured free cholesterol in these experiments.

Aortic Ring Neovascularization Assay

The method was adopted from reference³⁰, with modifications. Thoracic aorta was isolated from a 6-week old male C57BL6 mouse or an age and gender matched Abcg1^(−/−) mouse (kindly provided by Catherine Hedrick, La Jolla Institute for Allergy and Immunology), cleaned from surrounding fat and connective tissue and sliced into 1 mm long rings. The aortic rings were placed in wells of a 48-well plate containing solidified Matrigel and then covered with additional Matrigel and let it solidify for 30 min at 37° C. Small wells were made in Matrigel approximately 0.5 mm from aortic rings and 50 μl aliquots of Matrigel containing 1×10⁵ HEK293 cells transfected with mCherry (negative control), zAIBP2 or hAIBP were placed in these wells. After 10 min, each well was filled with EBM medium supplemented with 10 ng/ml VEGF and the plates were incubated at 37° C. for 6 days. Media were changed every two days. The rings were photographed in phase contrast using a Nikon Eclipse Ti microscope.

Visualization of Lipid Rafts with Cholera Toxin B

HUVEC were plated on glass coverslips and preincubated with 50 μg/ml HDL₃, 0.1 μg/ml hAIBP, or 50 μg/ml HDL₃+0.1 μg/ml hAIBP for 4 hours. Cells were washed once with medium before the addition of 1 μg/ml Alexa Fluor 594-labeled cholera toxin B (CTB, from Invitrogen). Cells were incubated for 15 min at 4° C., washed with PBS, and then incubated for 15 min at 4° C. with an anti-CTB antibody (EMD Chemicals) to crosslink CTB and lipid rafts. After washing with PBS, cells were fixed in 4% PFA for 20 min at 4° C., mounted with a PROLONG ANTIFADE KIT with DAPI (Invitrogen/Life Technologies) and images were captured with a Leica DM IRE2™ fluorescent microscope.

Cell Fractionation

Lipid rafts (light membrane fractions) were isolated using a detergent-free, discontinuous gradient ultracentrifugation method¹⁴. Briefly, HUVEC were washed twice with ice cold PBS and cells were scraped from the plate in 0.5 M sodium carbonate buffer (pH 11.0) containing a protease inhibitor cocktail (Sigma), homogenized and sonicated 3×10 sec. Samples were adjusted to 45% sucrose by adding a 90% sucrose solution in MBS (25 mM Mes, 0.15 M NaCl, pH 6.5) and placed into ultracentrifugation tubes. A 5-35% sucrose discontinuous gradient was formed above the sample, followed by ultracentrifugation at 35×10³ rpm for 18 hours at 4° C. in a SW-41 rotor (Beckman). Ten 1 ml fractions were collected from the top to the bottom of each gradient. The lipid rafts fraction (fraction 5) and the non-lipid rafts fraction (fraction 10) were used for further analysis, which included measurements of protein concentration and immunoblotting. Thirty μl of lipid rafts and non-lipid rafts fractions (adjusted to load equal protein concentrations of each sample) were run on SDS-PAGE, transferred to PVDF membranes and blotted with the indicated antibodies.

Caveolin-1 and VEGFR2 Colocalization

HUVEC plated on chamber coverglass (LAB-TEK II™) were incubated for 4 hours with 0.1 μg/ml hAIBP, 50 μg/ml HDL₃ or 0.1 μg/ml hAIBP+50 μg/ml HDL₃, and then cells were washed with PBS and fixed with warm 4% PFA for 15 min at room temperature. HUVEC were permeabilized with PBS containing 0.3% Triton X-100 (PBST) for 10 min, blocked with PBST containing 1% BSA and 5% normal goat serum for 1 hour at room temperature, incubated with anti-Caveolin-1 (BD Biosciences) and anti-VEGFR2 (Cell Signaling Technology) antibodies overnight at 4° C., followed by incubation with anti-mouse IgG-Alexa Fluor 488 and anti-rabbit IgG-Cy3 antibodies for 2 hours. Images were captured using a Nikon Eclipse Ti inverted fluorescent microscope operating in TIRF mode. Raw Tiff images of Caveolin-1 and VEGFR2 were analyzed using JACoP algorithm³¹. Colocalization was quantified using Pearson's coefficient. The JACoP plugin was loaded to ImageJ as described³¹.

VEGFR2 Endocytosis

HUVEC were incubated for 4 hours with 0.1 μg/ml hAIBP, 50 μg/ml HDL₃ or 0.1 μg/ml hAIBP+50 μg/ml HDL₃, followed by a 20 min incubation with 50 ng/ml VEGF. Cells were fixed and stained with antibodies against VEGFR2 and the early endosomal marker EEA-1-FITC (BD Biosciences). Images were captured with a Nikon Eclipse Ti inverted fluorescent microscope. VEGFR2 and EEA-1 colocalization was quantified using Pearson's coefficient with the JACoP plugin loaded to ImageJ³¹.

VEGFR2 Dimerization Assay

The assay was carried out as described in reference³². Two days after plating 1×10⁶HUVEC in a 10 cm dish, the cells were starved overnight in 0.5% FBS-EBM. Next day, cells were incubated for 4 hours with 0.2 μg/ml hAIBP, 50 μg/ml HDL₃ or 0.2 μg/ml hAIBP+50 μg/ml HDL₃ in EBM containing 0.2% BSA, followed by a 20 min incubation with 50 ng/ml VEGF and then crosslinked with 1 mg/ml bis-sulfosuccinimidyl (Thermo Scientific) for 30 min on ice. Cell lysates were immunoprecipitated with an anti-VEGFR2 antibody (Cell Signaling Technology) immobilized on agarose beads. The beads were washed and the eluted samples were run on SDS-PAGE, followed by immunoblotting with the VEGFR2 antibody.

HUVEC Migration Assay

Serum starved HUVEC were pretreated with 50 μg/ml HDL₃, 0.2 μg/ml hAIBP, or 50 μg/ml HDL₃+0.2 μg/ml hAIBP for 4 hours at 37° C. in 5% LPDS/EBM, harvested from the plate, washed, resuspended in 5% LPDS/EBM and added to the transwell (8 μm pore size). VEGF was added to the lower chamber at 20 ng/ml. Following a 4 hour incubation, the transwell membranes were fixed in ice-cold methanol for 10 min and stained with filtered 0.5% Crystal Violet for 10 min, and transmigrated cells were counted.

Zebrafish

Wild type AB and transgenic Tg(fli1:egfp)^(y1) and Tg(flk1:ras-cherry)^(s896) zebrafish lines^(19,33) were kindly provided by Dr. David Traver and Neil Chi (UCSD). Zebrafish were maintained as previously described³⁴, and all experimental procedures were approved by the UCSD IACUC.

Confocal Microscopy

Confocal imaging was carried out as previously described³⁵. Briefly, anaesthetized zebrafish embryos (treated at 24 hpf with 0.003% PTU) were housed in a sealed chamber (Invitrogen) in a small drop of 0.02% tricaine (Sigma) containing E3 medium. A Nikon C1-si confocal microscope was used in a one- or two-channel mode to detect fluorescence of one or two fluorophores in the same embryo. In the green channel, excitation was 488 nm for EGFP, and in the red channel, 561 nm for mCherry or DsRed. Z-stacks were acquired with a 1-3 μm step, and images were 3D rendered and analyzed using IMARIS® software (Bitplane). All 3D reconstructions were performed with the same threshold settings.

Morpholino Oligonucleotide Injections

To knock down gene expression, 4-8 ng of morpholino antisense oligonucleotides (MO; synthesized by GeneTools) were injected into one-cell stage embryos. A control MO was derived

from the zaibp2 sequence, with 5 mismatched oligonucleotides.

Control MO: (SEQ ID NO: 27) TGAGCTTCATGTTCATTTATTCCGC; zaibp2 MO (zaibp2 MO1): (SEQ ID NO: 28) TGTGGTTCATCTTGATTTATTCGGC; zaibp2 splicing MO (zaibp2 MO2): (SEQ ID NO: 29) TGTTGAGTGTCAGACAAACCTTGGT zaibp1 MO: (SEQ ID NO: 30) TCTGTATTCAAATCAGACGCTCAGT; abca1 MO: (SEQ ID NO: 31) AACCCAACTGAGTGGAGACAGCCAT; abcg1 MO: (SEQ ID NO: 32) AAAAGGCTGCCATGAGACATGCCAT.

Quantitative Analysis of Cell Sprouts in Segmental Arteries (SeA) and Subintestinal Veins (SW)

To determine changes in segmental artery cell sprouts in embryos injected with MO targeting zaibp2, zaibp1, abca1 and/or abcg1, we counted abnormal projections in 4 to 6 pairs of segmental arteries in adjacent somite boundaries in each zebrafish. For each set of injections, 15 embryos (i.e. 60-90 sprouts) were examined Values were expressed as a number of ectopic sprouts per SeA. To examine sprouts in SW, only the sprouts moving in the ventral direction out of the SIV were counted. Values were expressed as a number of ventral SW sprouts per zebrafish.

Measuring Membrane Lipid Order with Polarity-Sensitive Probe

The experiments were carried out as described in reference^(36,37). Briefly, live Tg(flk1:ras-cherry)^(s896) zebrafish embryos were incubated with 5 μM Laurdan (Invitrogen) at 28° C. for 30 min. The concentration of a Laurdan stock solution was measured using OD at 365 nm and an extinction coefficient of 19000 cm⁻¹M⁻¹. After incubation with Laurdan, embryos were incubated with E3 medium for additional 30 min, fixed in PFA for 4 hours at room temperature, deyolked, and embedded in 1% low melting temperature agarose for imaging. Images were captured with a Leica SP5 confocal/multiphoton system, using a water immersion 20× objective. The confocal mode was used to capture mCherry fluorescence and the multiphoton mode was used to capture Laurdan images (ex 800 nm, em 400-460 nm and 470-530 nm) in the same embryos. The multiphoton results were displayed as pseudocolored GP (a measure of the membrane lipid order) images, derived from Laurdan ratiometric measurements and using a ImageJ plug-in as described³⁶. The quantitative data were obtained by measuring GP values in the areas corresponding to tip cells (top ⅓ of the SeA length), stalk cells (bottom ⅔ of the SeA length) and the dorsal aorta in several individual, mCherry-masked z-sections. This method ensured that GP values were derived only from EC (e.g. from the areas where mCherry was in focus in each z-section). The GP images in FIG. 3e were composed each from four to five individual z-sections, with a minimal overlap of mCherry-masked GP images.

Whole Mount In Situ Hybridization

WISH was carried out as described³⁸⁻⁴⁰. Briefly, WT embryos or morphants at indicated developmental stages were fixed with 4% PFA and the embryos older than 24 hpf were permeabilized with 10 μg/ml protease K (Roche). Subsequently, the embryos were pre-hybridized at 70° C. for 4-6 hours, and then hybridized with a digoxigenin-labeled zaibp2 antisense probe at 65° C. for 2 days. Both the control sense and anti-sense RNA probes were directly synthesized from zaibp2 full length gene using a Roche T7/SP6 RNA or Ambion T3 RNA synthesis kit. After extensive wash, hybridized RNA was detected by immunohistochemistry using an alkaline phosphatase-conjugated antibody against digoxigenin (Roche) and a chromogenic substrate nitro blue tetrazolium (NBT) (Sigma) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)³⁸ (Sigma). A similar procedure was performed with tie2, vegfr2, vegfr3, fli1 and cdh5 probes. Double WISH were performed as described⁴¹. All the procedures were the same with the aforementioned one except that both digoxigenin-labeled zaibp2 and fluorescein-labeled myod were hybridized with the embryos. The embryos were then first incubated with alkaline phosphatase-conjugated anti-fluorescein Ab, and fast red (Roche) was used as a substrate. Subsequently, the embryos were washed, fixed with 4% PFA and incubated with anti-digoxigenin Ab conjugated alkaline phosphatase, and then NBT/BCIP was used as a chromogenic substrate.

Transplantation Experiments

Cell transplantation was performed as described³⁹, with different combinations of donors and recipients as indicated in FIG. 4a . Donor embryos were of the Tg(fli1: egfp)^(y1) origin, and recipient embryos were of the wild type origin. At the one-cell stage, donor Tg(fli1:egfp)^(y1) embryos were injected with rhodamine-labeled dextran (Mini-Ruby, Invitrogen) as a lineage tracer. At the sphere stage (approximately 4 hpf), embryos were dechorionated by 0.4 mg/ml pronase (Sigma) and transferred to agarose wells (Adaptive Science Tools, Worcester, Mass.). Approximately 20-40 cells from the margin of a donor embryo were transferred to the margin of a recipient embryo. The recipient embryos were subsequently grown at 28° C. and imaged at 72 hpf. Endothelial cells in chimeric zebrafish originating from donor embryos were visualized by their green fluorescence using a Leica M165FC fluorescent stereoscope. For detailed analysis, images were captured using a Nikon C1-si confocal microscope. Numbers of ectopic branches in each fluorescent SeA were counted.

Microarray

Fifty control MO or zaibp2 MO embryos at 12 hpf were dechorionated, and total RNA was extracted with a Qiagen's RNeasy Kit, with modifications. In brief, dechorionated embryos were immediately placed in RNAlater (Qiagen), and incubated for 10 min at room temperature. The embryos were then homogenized in Trizol (Invitrogen) for 30-60 seconds on ice, chloroform was added and the sample was vortexed for 30 sec, incubated for 5 min at room temperature and centrifuged for 30 min at 13,000 rpm at 4° C. The upper layer was transferred to a new tube, supplemented with 70% ethanol and mixed thoroughly. The sample was then processed according to the RNeasy kit manual. The microarray analysis was performed at UCSD's Biogem Core facility using a Zebrafish (V3) Gene Expression Microarray, 4×44k, Cat# G2519F-026437 from Agilent.

Real Time PCR

Real time PCR was performed using a Rotor Gene Q qPCR machine (Qiagen). Real time PCR master mix Platinum SYBR Green qPCR SUPERMIX was from Invitrogen (Life Technologies). The primers were synthesized by IDT. The PCR program was: 50° C. for 2 minutes (UDG incubation), 95° C. for 2 minutes, 40 cycles of: 95° C. for 15 seconds, 60° C. for 1 minute. Primer sequences:

fli1 (F): (SEQ ID NO: 33) CTTGGCACGTTGCCTTGATAAG, fli1 (R): (SEQ ID NO: 34) CCTTCATATCTGAGAGTGATCCC; tie2 (F): (SEQ ID NO: 35) GCGATGGATGGCAATAGAGT, tie2 (R): (SEQ ID NO: 36) CGACAGCAGGATCTGAGAGA; vegfr2 (F): (SEQ ID NO: 37) TCCACGAGGGTGGGCAGTCA; vegfr2 (R): (SEQ ID NO: 38) AGACGGGTGGTGTGGAGTAACGA. kdrb (F): (SEQ ID NO: 39) TGCCCACATGGAGCTGCTAGCA; kdrb (R): (SEQ ID NO: 40) TGTGGCACATTCAACCACATGAGC. β-actin (F): (SEQ ID NO: 41) CTCTTCCAGCCTTCCTTCCT, β-actin (R): (SEQ ID NO: 42) GGTTGGTTCGTTCGTTTGAAT.

Immunoblot of Zebrafish Lysates

Zebrafish were lysed on ice with a lysis buffer (50 mM Tris-HCl, pH 7.5, 4 mM sodium deoxycholate, 1% Triton X 100, 150 mM NaCl, 1 mM EDTA, and protease inhibitor cocktail from Sigma). Protein content was determined with a DC protein assay kit (BioRad) and equal protein amounts of the cell lysates were run on a 4-12% Bis-Tris SDS-PAGE with MOPS buffer (Invitrogen) and then transferred to a PVDF membrane (Invitrogen). The blots were probed with appropriate antibodies against specific phosphorylated and non-phosphorylated proteins (Cell Signaling Technology), secondary antibodies conjugated with HRP and developed using a Super Signal West Dura substrate (Pierce).

Filipin Staining

Filipin staining of embryos was performed as described⁴². Zebrafish were fixed with 4% PFA overnight at 4° C. The fixed fish were incubated overnight with 0.05% filipin (Sigma) in PBS with 1% sheep serum, and then washed 3 times with PBS. Images were captured immediately with a Leica M165FC fluorescent stereoscope and quantified.

Total Lipid Extraction and Free Cholesterol Measurements in Zebrafish Total lipid was extracted from zebrafish embryos as we previously described²⁸. In brief, trunk/tail segments were dissected from fifty 24 hpf embryos and pooled together. The tissue was homogenized and supplemented with 50 μg stigmasterol, an internal standard to control for recovery of extracted sterols. Total lipid extraction was performed with 1:2 methanol/dichloromethane. No saponification of cholesterol esters was performed because the goal of this study was to measure free cholesterol, the form of cholesterol transferred from the cells via ABC transporters to ApoA-VHDL. Cholesterol and stigmasterol were measured with a Shimadzu GC-2014 gas chromatograph using a m×0.25 mm (i d) ZB-5HT inferno capillary column, film thickness 0.2 nm (Phenomenex). Cholesterol levels were normalized to protein and then to the levels in embryos injected with control MO. References

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Example 4 Exemplary Compounds and Methods of the Invention are Effective for Treating or Ameliorating Cancer or Tumors

This example describes and demonstrates exemplary activities, and the efficacy, of compounds and methods of the invention; and in particular describes and demonstrates that compounds and methods of the invention can inhibit or slow or reverse the growth of a vascularized tumor, dysplastic tissue, tissue or growth, or cancer, using a method comprising administering to an individual an effective amount of a APOA1BP activity increasing formulation or an APOA1BP activity-increasing pharmaceutical composition of the invention.

FIG. 1 (Example 4) shows the tumor growth in mice injected with B16 melanoma cells. After tumor initiation, one group of mice received intravenous injections of recombinant human AIBP and the other group received injections of a control protein. AIBP inhibited tumor growth.

FIG. 2 (Example 4) shows the white blood cell differential in mice fed a high-fed diet (HFD) for 4 weeks. It has been documented that the HFD induces myeloid cell (monocytes and neutrophils) proliferation, reflective of an inflammatory status of hyperlipidemic animals. Injections of human AIBP but not of a control protein reversed myelocytosis in these animals.

These results demonstrate that AIBP selectively inhibit proliferation of myeloid progenitor cells, and thus can be used for treatment of dysplastic tissue, tissue or growth, or cancer, including myeloid cell proliferation in patients with cardiovascular disease, diabetes and in the aging population, as well as for treatment of myeloproliferative disease.

While data described herein links the therapeutic effects of AIBP on angiogenesis and cancer and myeloproliferative conditions (FIGS. 1 and 2, Example 4) to its ability to promote cholesterol removal from endothelial cells, tumor and/or tumor-associated cells, and myeloid progenitor cells, respectively, the invention is not limited by any particular mechanism of action.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims. 

What is claimed is:
 1. A formulation or a pharmaceutical composition comprising: (a) an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP) polypeptide compound or composition, or a compound that increases expression or activity of a APOA1BP polypeptide or nucleic acid, or an a APOA1BP polypeptide or peptide having a APOA1BP activity; (b) the formulation or pharmaceutical composition of (d), wherein the compound that increases expression or activity of a APOA1BP polypeptide is a nucleic acid that expresses a APOA1BP polypeptide or a polypeptide having a APOA1BP polypeptide activity, and optionally a APOA1BP-stimulating compound or composition increases or stimulates (activates) the activity of a APOA1BP promoter or transcriptional regulatory sequence or motif; (c) an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP)-inhibiting compound or composition; (d) the formulation or pharmaceutical composition of (a), wherein the APOA1BP-inhibiting compound or composition inhibits or decreases the expression of a APOA1BP nucleic acid, wherein optionally the nucleic acid inhibited is a APOA1BP gene or a APOA1BP message (mRNA), and optionally the APOA1BP-inhibiting nucleic acid is an APOA1BP antisense oligonucleotide, or a APOA1BP-inhibiting miRNA or siRNA and optionally a APOA1BP-inhibiting compound or composition inhibits or decreases the activity of a APOA1BP promoter or transcriptional regulatory sequence or motif; (e) the formulation or pharmaceutical composition of (a), wherein the APOA1BP-inhibiting compound or composition inhibits or decreases the expression of a APOA1BP polypeptide, wherein optionally the APOA1BP polypeptide-inhibiting compound is an anti-APOA1BP antibody or a APOA1BP-binding antibody or antibody-like fragment, a small molecule, a lipid or a carbohydrate; (f) the formulation or pharmaceutical composition of any of (a) to (e), wherein the an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP) polypeptide or protein is a human or a mammalian APOA1BP, or a AIBP1 or a AIBP2, or a recombinant, peptidomimetic or a synthetic APOA1BP, or a bioisostere of an ApoA-I Binding Protein (APOA1BP, AIBP, or AI-BP); (g) the formulation or pharmaceutical composition of any of (a) to (f), wherein the APOA1BP-inhibiting compounds comprise an antisense morpholino oligonucleotide; or (h) the formulation or pharmaceutical composition of any of (a) to (f), wherein the APOA1BP-inhibiting or APOA1BP-stimulating compound or composition is formulated for administration in vivo; or for enteral or parenteral administration, or as a liposome, a tablet, a pill, a capsule, a gel, a geltab, a liquid, a powder, an emulsion, a lotion, an aerosol, a spray, a lozenge, an aqueous or a sterile or an injectable solution, or an implant.
 2. A method for stimulating cholesterol efflux from a cell, comprising contacting the cell with an effective amount of a APOA1BP activity increasing formulation or a pharmaceutical composition of claim
 1. 3. A method for inhibiting, reversing or reducing the rate of formation of lipid rafts or cholesterol-/sphingolipids membrane microdomains, or increasing levels of high density lipoprotein (HDL) and/or improving HDL function, comprising administering to an individual an effective amount of an APOA1BP activity-increasing APOA1BP activity increasing formulation or a pharmaceutical composition of claim
 1. 4. A method for inhibiting or slowing vascular endothelial cell growth, or neovascularization, or angiogenesis, comprising administering to an individual an effective amount of a APOA1BP activity increasing formulation or an APOA1BP activity-increasing pharmaceutical composition of claim
 1. 5. A method for inhibiting or slowing or reversing the growth of a vascularized tumor, dysplastic tissue, tissue or growth, or cancer, comprising administering to an individual an effective amount of a APOA1BP activity increasing formulation or an APOA1BP activity-increasing pharmaceutical composition of claim
 1. 6. A method for treating, preventing, ameliorating or reversing the effects of a dyslipidemia, hyperlipidemia, atherosclerosis or coronary artery disease, comprising administering to an individual an effective amount of a APOA1BP activity increasing formulation or an APOA1BP activity-increasing pharmaceutical composition of claim
 1. 7. A method for promoting or accelerating revascularization, or promoting revascularization of the heart, or promoting revascularization of the heart following an ischemic event or promoting post-ischemic vascularization, comprising administering a APOA1BP-inhibiting formulation or an APOA1BP activity-inhibiting pharmaceutical composition of claim
 1. 8. A method for interfering with vascular endothelial growth factor receptor 2 (VEGRF2) (also known as kinase insert domain receptor, or KDR) dimerization or signaling, comprising administering to an individual an effective amount of a APOA1BP activity increasing formulation or an APOA1BP activity-increasing pharmaceutical composition of claim
 1. 9. A kit comprising a compound, composition or formulation of claim
 1. 10. A nanoparticle, nanolipoparticle, vesicle or liposomal membrane comprising a compound, composition or formulation of claim
 1. 11-18. (canceled)
 19. The method of claim 2, wherein the formulation or pharmaceutical composition comprises a APOA1BP polypeptide or peptide having APOA1BP activity, or a APOA1BP-stimulating polypeptide compound or composition, or a compound that increases expression or activity of a APOA1BP polypeptide or nucleic acid, wherein optionally the administration is in vitro, ex vivo or in vivo. and optionally the cell is an endothelial cell, or a human cell.
 20. The method of claim 3, wherein the formulation or pharmaceutical composition comprises a APOA1BP polypeptide or peptide having APOA1BP activity, a APOA1BP-stimulating polypeptide compound or composition, or a compound that increases expression or activity of a APOA1BP polypeptide or nucleic acid, wherein optionally the administration is ex vivo or in vivo, and optionally the individual is a human.
 21. The method of claim 4, wherein the formulation or pharmaceutical composition comprises a APOA1BP polypeptide or peptide having APOA1BP activity, a APOA1BP-stimulating polypeptide compound or composition, or a compound that increases expression or activity of a APOA1BP polypeptide or nucleic acid, wherein optionally the administration is ex vivo or in vivo, and optionally the individual is a human.
 22. The method of claim 5, wherein the formulation or pharmaceutical composition comprises a APOA1BP polypeptide or peptide having APOA1BP activity, a APOA1BP-stimulating polypeptide compound or composition, or a compound that increases expression or activity of a APOA1BP polypeptide or nucleic acid, wherein optionally the administration is ex vivo or in vivo, and optionally the individual is a human.
 23. The method of claim 6, wherein the formulation or pharmaceutical composition comprises a APOA1BP polypeptide or peptide having APOA1BP activity, a APOA1BP-stimulating polypeptide compound or composition, or a compound that increases expression or activity of a APOA1BP polypeptide or nucleic acid, wherein optionally the administration is ex vivo or in vivo, and optionally the individual is a human.
 24. The method of claim 7, wherein: the APOA1BP-inhibiting compound or composition inhibits or decreases the expression of a APOA1BP nucleic acid, wherein optionally the nucleic acid inhibited is a APOA1BP gene or a APOA1BP message (mRNA), and optionally the APOA1BP-inhibiting nucleic acid is an APOA1BP antisense oligonucleotide, or a APOA1BP-inhibiting miRNA or siRNA and optionally a APOA1BP-inhibiting compound or composition inhibits or decreases the activity of a APOA1BP promoter or transcriptional regulatory sequence or motif; or and the APOA1BP-inhibiting compound or composition inhibits or decreases the expression of a APOA1BP polypeptide, wherein optionally the APOA1BP polypeptide-inhibiting compound is an anti-APOA1BP antibody or a APOA1BP-binding antibody or antibody-like fragment, a small molecule, a lipid or a carbohydrate.
 25. The method of claim 8, wherein: the formulation or pharmaceutical composition comprises a APOA1BP polypeptide or peptide having APOA1BP activity, a APOA1BP-stimulating polypeptide compound or composition, or a compound that increases expression or activity of a APOA1BP polypeptide or nucleic acid, wherein optionally the administration is in vitro, ex vivo or in vivo, and optionally the individual is a human. 